Methods for producing hyaluronan in a recombinant host cell

ABSTRACT

The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase

CROSS-REFERENCES TO RELATED APPLICATION

[0001] This application claims priority from U.S. provisional patentapplication Ser. No. 60/342,644 filed Dec. 21, 2001, which applicationis fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for producing ahyaluronan in a recombinant host cell.

[0004] 2. Description of the Related Art

[0005] The most abundant heteropolysaccharides of the body are theglycosaminoglycans. Glycosaminoglycans are unbranched carbohydratepolymers, consisting of repeating disaccharide units (only keratansulphate is branched in the core region of the carbohydrate). Thedisaccharide units generally comprise, as a first saccharide unit, oneof two modified sugars —N-acetylgalactosamine (GalNAc) orN-acetylglucosamine (GlcNAc). The second unit is usually an uronic acid,such as glucuronic acid (GlcUA) or iduronate.

[0006] Glycosaminoglycans are negatively charged molecules, and have anextended conformation that imparts high viscosity when in solution.Glycosaminoglycans are located primarily on the surface of cells or inthe extracellular matrix. Glycosaminoglycans also have lowcompressibility in solution and, as a result, are ideal as aphysiological lubricating fluid, e.g., joints. The rigidity ofglycosaminoglycans provides structural integrity to cells and providespassageways between cells, allowing for cell migration. Theglycosaminoglycans of highest physiological importance are hyaluronan,chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, andkeratan sulfate. Most glycosaminoglycans bind covalently to aproteoglycan core protein through specific oligosaccharide structures.Hyaluronan forms large aggregates with certain proteoglycans, but is anexception as free carbohydrate chains form non-covalent complexes withproteoglycans.

[0007] Numerous roles of hyaluronan in the body have been identified(see, Laurent T. C. and Fraser J. R. E., 1992, FASEB J. 6: 2397-2404;and Toole B. P., 1991, “Proteoglycans and hyaluronan in morphogenesisand differentiation.” In: Cell Biology of the Extracellular Matrix, pp.305-341, Hay E. D., ed., Plenum, N.Y.). Hyaluronan is present in hyalinecartilage, synovial joint fluid, and skin tissue, both dermis andepidermis. Hyaluronan is also suspected of having a role in numerousphysiological functions, such as adhesion, development, cell motility,cancer, angiogenesis, and wound healing. Due to the unique physical andbiological properties of hyaluronan, it is employed in eye and jointsurgery and is being evaluated in other medical procedures. Products ofhyaluronan have also been developed for use in orthopaedics,rheumatology, and dermatology.

[0008] Rooster combs are a significant commercial source for hyaluronan.Microorganisms are an alternative source. U.S. Pat. No. 4,801,539discloses a fermentation method for preparing hyaluronic acid involvinga strain of Streptococcus zooepidemicus with reported yields of about3.6 g of hyaluronic acid per liter. European Patent No. EP0694616discloses fermentation processes using an improved strain ofStreptococcus zooepidemicus with reported yields of about 3.5 g ofhyaluronic acid per liter.

[0009] The microorganisms used for production of hyaluronic acid byfermentation are strains of pathogenic bacteria, foremost among thembeing several Streptococcus spp. The group A and group C streptococcisurround themselves with a nonantigenic capsule composed of hyaluronan,which is identical in composition to that found in connective tissue andjoints. Pasteurella multocida, another pathogenic encapsulatingbacteria, also surrounds its cells with hyaluronan.

[0010] Hyaluronan synthases have been described from vertebrates,bacterial pathogens, and algal viruses (DeAngelis, P. L., 1999, Cell.Mol. Life Sci. 56: 670-682). WO 99/23227 discloses a Group I hyaluronatesynthase from Streptococcus equisimilis. WO 99/51265 and WO 00/27437describe a Group II hyaluronate synthase from Pasturella multocida.Ferretti et al. disclose the hyaluronan synthase operon of Streptococcuspyogenes, which is composed of three genes, hasA, hasB, and hasC, thatencode hyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucosepyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98,4658-4663, 2001). WO 99/51265 describes a nucleic acid segment having acoding region for a Streptococcus equisimilis hyaluronan synthase.

[0011] Bacilli are well established as host cell systems for theproduction of native and recombinant proteins. It is an object of thepresent invention to provide methods for producing a hyaluronan in arecombinant Bacillus host cell.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention relates to methods for producing ahyaluronic acid, comprising: (a) cultivating a Bacillus host cell underconditions suitable for production of the hyaluronic acid, wherein theBacillus host cell comprises a nucleic acid construct comprising ahyaluronan synthase encoding sequence operably linked to a promotersequence foreign to the hyaluronan synthase encoding sequence; and (b)recovering the hyaluronic acid from the cultivation medium.

[0013] In preferred embodiments, the nucleic acid construct furthercomprises one or more genes encoding enzymes in the biosynthesis of aprecursor sugar of the hyaluronic acid or the Bacillus host cell furthercomprises one or more second nucleic acid constructs comprising one ormore genes encoding enzymes in the biosynthesis of the precursor sugar.

[0014] In another preferred embodiment, the one or more genes encoding aprecursor sugar are under the control of the same or a differentpromoter(s) as the hyaluronan synthase encoding sequence.

[0015] The present invention also relates to Bacillus host cellscomprising a nucleic acid construct comprising a hyaluronan synthaseencoding sequence operably linked to a promoter sequence foreign to thehyaluronan synthase encoding sequence, and to such nucleic acidconstructs.

[0016] The present invention also relates to an isolated nucleic acidsequence encoding a hyaluronan synthase operon comprising a hyaluronansynthase gene or a portion thereof and a UDP-glucose 6-dehydrogenasegene, and optionally one or more genes selected from the groupconsisting of a UDP-glucose pyrophosphorylase gene,UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphateisomerase gene.

[0017] The present invention also relates to isolated nucleic acidsequences encoding a UDP-glucose 6-dehydrogenase selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least about 75%, about 80%, about85%, about 90%, or about 95% identity to SEQ ID NO: 41; (b) a nucleicacid sequence having at least about 75%, about 80%, about 85%, about90%, or about 95% homology to SEQ ID NO: 40; (c) a nucleic acid sequencewhich hybridizes under medium or high stringency conditions with (i) thenucleic acid sequence of SEQ ID NO: 40, (ii) the cDNA sequence containedin SEQ ID NO: 40, or (iii) a complementary strand of (i) or (ii); and(d) a subsequence of (a), (b), or (c), wherein the subsequence encodes apolypeptide fragment which has UDP-glucose 6-dehydrogenase activity.

[0018] The present invention also relates to isolated nucleic acidsequences encoding a UDP-glucose pyrophosphorylase selected from thegroup consisting of: (a) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence which has at least about 90%, about 95%,or about 97% identity to SEQ ID NO: 43; (b) a nucleic acid sequencehaving at least about 90%, about 95%, or about 97% homology to SEQ IDNO: 42; (c) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with (i) the nucleic acid sequence of SEQID NO: 42, (ii) the cDNA sequence contained in SEQ ID NO: 42, or (iii) acomplementary strand of (i) or (ii); and (d) a subsequence of (a), (b),or (c), wherein the subsequence encodes a polypeptide fragment which hasUDP-N-acetylglucosamine pyrophosphorylase activity.

[0019] The present invention also relates to isolated nucleic acidsequences encoding a UDP-N-acetylglucosamine pyrophosphorylase selectedfrom the group consisting of: (a) a nucleic acid sequence encoding apolypeptide having an amino acid sequence which has at least about 75%,about 80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 45;(b) a nucleic acid sequence having at least about 75%, about 80%, about85%, about 90%, or about 95% homology to SEQ ID NO: 44; (c) a nucleicacid sequence which hybridizes under low, medium, or high stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 44, (ii) thecDNA sequence contained in SEQ ID NO: 44, or (iii) a complementarystrand of (i) or (ii); and (d) a subsequence of (a), (b), or (c),wherein the subsequence encodes a polypeptide fragment which hasUDP-N-acetylglucosamine pyrophosphorylase activity.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows the chemical structure of hyaluronan.

[0021]FIG. 2 shows the biosynthetic pathway for hyaluronan synthesis.

[0022]FIG. 3 shows a restriction map of pCR2.1-sehasA.

[0023]FIG. 4 shows a restriction map of pCR2.1-tuaD.

[0024]FIG. 5 shows a restriction map of pCR2.1-gtaB.

[0025]FIG. 6 shows a restriction map of pCR2.1-gcaD.

[0026]FIG. 7 shows a restriction map of pHA1.

[0027]FIG. 8 shows a restriction map of pHA2.

[0028]FIG. 9 shows a restriction map of pHA3.

[0029]FIG. 10 shows a restriction map of pHA4.

[0030]FIG. 11 shows a restriction map of pHA5.

[0031]FIG. 12 shows a restriction map of pHA6.

[0032]FIG. 13 shows a restriction map of pHA7.

[0033]FIG. 14 shows a restriction map of pMRT106.

[0034]FIG. 15 shows a restriction map of pHA8.

[0035]FIG. 16 shows a restriction map of pHA9.

[0036]FIG. 17 shows a restriction map of pHA10.

[0037]FIG. 18 shows a restriction map of pRB157.

[0038]FIG. 19 shows a restriction map of pMRT084.

[0039]FIG. 20 shows a restriction map of pMRT086.

[0040]FIG. 21 shows a restriction map of pCJ791.

[0041]FIG. 22 shows a restriction map of pMRT032.

[0042]FIG. 23 shows a restriction map of pNNB194neo.

[0043]FIG. 24 shows a restriction map of pNNB194neo-oriT.

[0044]FIG. 25 shows a restriction map of pShV3.

[0045]FIG. 26 shows a restriction map of pShV2.1-amyEΔB.

[0046]FIG. 27 shows a restriction map of pShV3A.

[0047]FIG. 28 shows a restriction map of pMRT036.

[0048]FIG. 29 shows a restriction map of pMRT037.

[0049]FIG. 30 shows a restriction map of pMRT041.

[0050]FIG. 31 shows a restriction map of pMRT064.1.

[0051]FIG. 32 shows a restriction map of pMRT068.

[0052]FIG. 33 shows a restriction map of pMRT069.

[0053]FIG. 34 shows a restriction map of pMRT071.

[0054]FIG. 35 shows a restriction map of pMRT074.

[0055]FIG. 36 shows a restriction map of pMRT120.

[0056]FIG. 37 shows a restriction map of pMRT122.

[0057]FIG. 38 shows a restriction map of pCR2.1-pel5′.

[0058]FIG. 39 shows a restriction map of pCR2.1-pel3′.

[0059]FIG. 40 shows a restriction map of pRB161.

[0060]FIG. 41 shows a restriction map of pRB162.

[0061]FIG. 42 shows a restriction map of pRB156.

[0062]FIG. 43 shows a restriction map of pRB164.

[0063]FIG. 44 shows a summary of fermentations of various hyaluronicacid producing Bacillus subtilis strains run under fed batch atapproximately 2 g sucrose/L₀-hr, 37° C.

[0064]FIG. 45 shows a summary of peak hyaluronic acid weight averagemolecular weights (MDa) obtained from fermentations of varioushyaluronic acid producing Bacillus subtilis strains run under fed batchat approximately 2 g sucrose/L₀-hr, 37° C.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention relates to methods for producing ahyaluronan, comprising: (a) cultivating a Bacillus host cell underconditions suitable for production of the hyaluronan, wherein theBacillus host cell comprises a nucleic acid construct comprising ahyaluronan synthase encoding sequence operably linked to a promotersequence foreign to the hyaluronan synthase encoding sequence; and (b)recovering the hyaluronan from the cultivation medium.

[0066] The methods of the present invention represent an improvementover the production of hyaluronan from pathogenic, encapsulatingbacteria. In encapsulating bacteria, a large quantity of the hyaluronanis produced in the capsule. In processing and purifying hyaluronan fromsuch sources, it is first necessary to remove the hyaluronan from thecapsule, such as by the use of a surfactant, or detergent, such as SDS.This creates a complicating step in commercial production of hyaluronan,as the surfactant must be added in order to liberate a large portion ofthe hyaluronan, and subsequently the surfactant must be removed prior tofinal purification.

[0067] The present invention allows the production of a large quantityof a hyaluronan, which is produced in a non-encapsulating host cell, asfree hyaluronan. When viewed under the microscope, there is no visiblecapsule associated with the recombinant strains of Bacillus, whereas thepathogenic strains traditionally used in hyaluronan production comprisea capsule of hyaluronan that is at least twice the diameter of the cellitself.

[0068] Since the hyaluronan of the recombinant Bacillus cell isexpressed directly to the culture medium, a simple process may be usedto isolate the hyaluronan from the culture medium. First, the Bacilluscells and cellular debris are physically removed from the culturemedium. The culture medium may be diluted first, if desired, to reducethe viscosity of the medium. Many methods are known to those skilled inthe art for removing cells from culture medium, such as centrifugationor microfiltration. If desired, the remaining supernatant may then befiltered, such as by ultrafiltration, to concentrate and remove smallmolecule contaminants from the hyaluronan. Following removal of thecells and cellular debris, a simple precipitation of the hyaluronan fromthe medium is performed by known mechanisms. Salt, alcohol, orcombinations of salt and alcohol may be used to precipitate thehyaluronan from the filtrate. Once reduced to a precipitate, thehyaluronan can be easily isolated from the solution by physical means.Alternatively, the hyaluronan may be dried or concentrated from thefiltrate solution by using evaporative techniques known to the art, suchas spray drying.

[0069] The methods of the present invention thus represent animprovement over existing techniques for commercially producinghyaluronan by fermentation, in not requiring the use of a surfactant inthe purification of hyaluronan from cells in culture.

[0070] Hyaluronic Acid

[0071] “Hyaluronic acid” is defined herein as an unsulphatedglycosaminoglycan composed of repeating disaccharide units ofN-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked togetherby alternating beta-1,4 and beta-1,3 glycosidic bonds (FIG. 1).Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. Theterms hyaluronan and hyaluronic acid are used interchangeably herein.

[0072] In a preferred embodiment, the hyaluronic acid obtained by themethods of the present invention has a molecular weight of about 10,000to about 10,000,000 Da. In a more preferred embodiment, the hyaluronicacid obtained by the methods of the present invention has a molecularweight of about 25,000 to about 5,000,000 Da. In a most preferredembodiment, the hyaluronic acid obtained by the methods of the presentinvention has a molecular weight of about 50,000 to about 3,000,000 Da.

[0073] The level of hyaluronic acid produced by a Bacillus host cell ofthe present invention may be determined according to the modifiedcarbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).Moreover, the average molecular weight of the hyaluronic acid may bedetermined using standard methods in the art, such as those described byUeno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal.Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, “Light ScatteringUniversity DAWN Course Manual” and “DAWN EOS Manual” Wyatt TechnologyCorporation, Santa Barbara, Calif.

[0074] The hyaluronic acid obtained by the methods of the presentinvention may be subjected to various techniques known in the art tomodify the hyaluronic acid, such as crosslinking as described, forexample, in U.S. Pat. Nos. 5,616,568, 5,652,347, and 5,874,417.Moreover, the molecular weight of the hyaluronic acid may be alteredusing techniques known in the art.

[0075] Host Cells

[0076] In the methods of the present invention, the Bacillus host cellmay be any Bacillus cell suitable for recombinant production ofhyaluronic acid. The Bacillus host cell may be a wild-type Bacillus cellor a mutant thereof. Bacillus cells useful in the practice of thepresent invention include, but are not limited to, Bacillusagaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.Mutant Bacillus subtilis cells particularly adapted for recombinantexpression are described in WO 98/22598. Non-encapsulating Bacilluscells are particularly useful in the present invention.

[0077] In a preferred embodiment, the Bacillus host cell is a Bacillusamyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. Ina more preferred embodiment, the Bacillus cell is a Bacillusamyloliquefaciens cell. In another more preferred embodiment, theBacillus cell is a Bacillus clausii cell. In another more preferredembodiment, the Bacillus cell is a Bacillus lentus cell. In another morepreferred embodiment, the Bacillus cell is a Bacillus licheniformiscell. In another more preferred embodiment, the Bacillus cell is aBacillus subtilis cell. In a most preferred embodiment, the Bacillushost cell is Bacillus subtilis A164 Δ5 (see U.S. Pat. No. 5,891,701) orBacillus subtilis 168 Δ4.

[0078] Transformation of the Bacillus host cell with a nucleic acidconstruct of the present invention may, for instance, be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, MolecularGeneral Genetics 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnauand Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221),by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987,Journal of Bacteriology 169: 5271-5278).

[0079] Nucleic Acid Constructs

[0080] “Nucleic acid construct” is defined herein as a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acid which are combined and juxtaposed in a manner whichwould not otherwise exist in nature. The term nucleic acid construct maybe synonymous with the term expression cassette when the nucleic acidconstruct contains all the control sequences required for expression ofa coding sequence. The term “coding sequence” is defined herein as asequence which is transcribed into mRNA and translated into an enzyme ofinterest when placed under the control of the below mentioned controlsequences. The boundaries of the coding sequence are generallydetermined by a ribosome binding site located just upstream of the openreading frame at the 5′ end of the mRNA and a transcription terminatorsequence located just downstream of the open reading frame at the 3′ endof the mRNA. A coding sequence can include, but is not limited to, DNA,cDNA, and recombinant nucleic acid sequences.

[0081] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are well known in the art and include, forexample, isolation from genomic DNA, preparation from cDNA, or acombination thereof. The cloning of the nucleic acid sequences from suchgenomic DNA can be effected, e.g., by using antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features or the well known polymerase chain reaction (PCR).See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methodsand Application, Academic Press, N.Y. Other nucleic acid amplificationprocedures such as ligase chain reaction, ligated activatedtranscription, and nucleic acid sequence-based amplification may beused. The cloning procedures may involve excision and isolation of adesired nucleic acid fragment comprising the nucleic acid sequenceencoding the polypeptide, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into a Bacilluscell where clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semi-synthetic,synthetic origin, or any combinations thereof.

[0082] An isolated nucleic acid sequence encoding an enzyme may bemanipulated in a variety of ways to provide for expression of theenzyme. Manipulation of the nucleic acid sequence prior to its insertioninto a construct or vector may be desirable or necessary depending onthe expression vector or Bacillus host cell. The techniques formodifying nucleic acid sequences utilizing cloning methods are wellknown in the art. It will be understood that the nucleic acid sequencemay also be manipulated in vivo in the host cell using methods wellknown in the art.

[0083] A number of enzymes are involved in the biosynthesis ofhyaluronic acid. These enzymes include hyaluronan synthase, UDP-glucose6-dehydrogenase, UDP-glucose pyrophosphorylase, UDP-N-acetylglucosaminepyrophosphorylase, glucose-6-phosphate isomerase, hexokinase,phosphoglucomutase, amidotransferase, mutase, and acetyl transferase.Hyaluronan synthase is the key enzyme in the production of hyaluronicacid.

[0084] “Hyaluronan synthase” is defined herein as a synthase thatcatalyzes the elongation of a hyaluronan chain by the addition of GlcUAand GlcNAc sugar precursors. The amino acid sequences of streptococcalhyaluronan synthases, vertebrate hyaluronan synthases, and the viralhyaluronan synthase are distinct from the Pasteurella hyaluronansynthase, and have been proposed for classification as Group I and GroupII hyaluronan synthases, the Group I hyaluronan synthases includingStreptococcal hyaluronan synthases (DeAngelis, 1999). For production ofhyaluronan in Bacillus host cells, hyaluronan synthases of a eukaryoticorigin, such as mammalian hyaluronan synthases, are less preferred.

[0085] The hyaluronan synthase encoding sequence may be any nucleic acidsequence capable of being expressed in a Bacillus host cell. The nucleicacid sequence may be of any origin. Preferred hyaluronan synthase genesinclude any of either Group I or Group II, such as the Group Ihyaluronan synthase genes from Streptococcus equisimilis, Streptococcuspyogenes, Streptococcus uberis, and Streptococcus equi subsp.zooepidemicus, or the Group II hyaluronan synthase genes of Pasturellamultocida.

[0086] In a preferred embodiment, the hyaluronan synthase encodingsequence is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 1, SEQ ID NO: 92, or SEQ ID NO:102; and (c) a complementary strand of (a) or (b).

[0087] In a more preferred embodiment, the hyaluronan synthase encodingsequence encodes a polypeptide having the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; or a fragment thereof havinghyaluronan synthase activity.

[0088] In another preferred embodiment, the hyaluronan synthase encodingsequence is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 95; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 94; and (c) a complementary strand of (a) or (b).

[0089] In another more preferred embodiment, the hyaluronan synthaseencoding sequence encodes a polypeptide having the amino acid sequenceof SEQ ID NO: 95, or a fragment thereof having hyaluronan synthaseactivity.

[0090] The methods of the present invention also include constructswhereby precursor sugars of hyaluronan are supplied to the host cell,either to the culture medium, or by being encoded by endogenous genes,by non-endogenous genes, or by a combination of endogenous andnon-endogenous genes in the Bacillus host cell. The precursor sugar maybe D-glucuronic acid or N-acetyl-glucosamine.

[0091] In the methods of the present invention, the nucleic acidconstruct may further comprise one or more genes encoding enzymes in thebiosynthesis of a precursor sugar of a hyaluronan. Alternatively, theBacillus host cell may further comprise one or more second nucleic acidconstructs comprising one or more genes encoding enzymes in thebiosynthesis of the precursor sugar. Hyaluronan production is improvedby the use of constructs with a nucleic acid sequence or sequencesencoding a gene or genes directing a step in the synthesis pathway ofthe precursor sugar of hyaluronan. By, “directing a step in thesynthesis pathway of a precursor sugar of hyaluronan” is meant that theexpressed protein of the gene is active in the formation ofN-acetyl-glucosamine or D-glucuronic acid, or a sugar that is aprecursor of either of N-acetyl-glucosamine and D-glucuronic acid (FIG.2).

[0092] In a preferred method for supplying precursor sugars, constructsare provided for improving hyaluronan production in a host cell having ahyaluronan synthase, by culturing a host cell having a recombinantconstruct with a heterologous promoter region operably linked to anucleic acid sequence encoding a gene directing a step in the synthesispathway of a precursor sugar of hyaluronan. In a preferred method thehost cell also comprises a recombinant construct having a promoterregion operably linked to a hyaluronan synthase, which may use the sameor a different promoter region than the nucleic acid sequence to asynthase involved in the biosynthesis of N-acetyl-glucosamine. In afurther preferred embodiment, the host cell may have a recombinantconstruct with a promoter region operably linked to different nucleicacid sequences encoding a second gene involved in the synthesis of aprecursor sugar of hyaluronan.

[0093] Thus, the present invention also relates to constructs forimproving hyaluronan production by the use of constructs with a nucleicacid sequence encoding a gene directing a step in the synthesis pathwayof a precursor sugar of hyaluronan. The nucleic acid sequence to theprecursor sugar may be expressed from the same or a different promoteras the nucleic acid sequence encoding the hyaluronan synthase.

[0094] The genes involved in the biosynthesis of precursor sugars forthe production of hyaluronic acid include a UDP-glucose 6-dehydrogenasegene, UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene.

[0095] In a cell containing a hyaluronan synthase, any one orcombination of two or more of hasB, hasC and hasD, or the homologsthereof, such as the Bacillus subtilis tuaD, gtaB, and gcaD,respectively, as well as hasE, may be expressed to increase the pools ofprecursor sugars available to the hyaluronan synthase. The Bacillusgenome is described in Kunst, et al., Nature 390, 249-256, “The completegenome sequence of the Gram-positive bacterium Bacillus subtilis” (Nov.20, 1997). In some instances, such as where the host cell does not havea native hyaluronan synthase activity, the construct may include thehasA gene.

[0096] The nucleic acid sequence encoding the biosynthetic enzymes maybe native to the host cell, while in other cases heterologous sequencemay be utilized. If two or more genes are expressed they may be genesthat are associated with one another in a native operon, such as thegenes of the HAS operon of Streptococcus equisimilis, which compriseshasA, hasB, hasC and hasD. In other instances, the use of somecombination of the precursor gene sequences may be desired, without eachelement of the operon included. The use of some genes native to the hostcell, and others which are exogenous may also be preferred in othercases. The choice will depend on the available pools of sugars in agiven host cell, the ability of the cell to accommodate overproductionwithout interfering with other functions of the host cell, and whetherthe cell regulates expression from its native genes differently thanexogenous genes.

[0097] As one example, depending on the metabolic requirements andgrowth conditions of the cell, and the available precursor sugar pools,it may be desirable to increase the production of N-acetyl-glucosamineby expression of a nucleic acid sequence encodingUDP-N-acetylglucosamine pyrophosphorylase, such as the hasD gene, theBacillus gcaD gene, and homologs thereof. Alternatively, the precursorsugar may be D-glucuronic acid. In one such embodiment, the nucleic acidsequence encodes UDP-glucose 6-dehydrogenase. Such nucleic acidsequences include the Bacillus tuaD gene, the hasB gene ofStreptococcus, and homologs thereof. The nucleic acid sequence may alsoencode UDP-glucose pyrophosphorylase, such as in the Bacillus gtaB gene,the hasC gene of Streptococcus, and homologs thereof.

[0098] In the methods of the present invention, the UDP-glucose6-dehydrogenase gene may be a hasB gene or tuaD gene; or homologsthereof.

[0099] In a preferred embodiment, the hasB gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 41, SEQID NO: 97, or SEQ ID NO: 105; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 40, SEQ ID NO: 96, or SEQ ID NO: 104; and (c) a complementary strandof (a) or (b).

[0100] In a more preferred embodiment, the hasB gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 41, SEQ ID NO:97, or SEQ ID NO: 105; or a fragment thereof having UDP-glucose6-dehydrogenase activity.

[0101] In another preferred embodiment, the tuaD gene is selected fromthe group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 12; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 11; and (c) acomplementary strand of (a) or (b).

[0102] In another more preferred embodiment, the tuaD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 12, or afragment thereof having UDP-glucose 6-dehydrogenase activity.

[0103] In the methods of the present invention, the UDP-glucosepyrophosphorylase gene may be a hasC gene or gtaB gene; or homologsthereof.

[0104] In a preferred embodiment, the hasC gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 43, SEQID NO: 99, or SEQ ID NO: 107; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 42 or SEQ ID NO: 98, or SEQ ID NO: 106; and (c) a complementarystrand of (a) or (b).

[0105] In another more preferred embodiment, the hasC gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 43 or SEQ IDNO: 99, or SEQ ID NO: 107; or a fragment thereof having UDP-glucosepyrophosphorylase activity.

[0106] In another preferred embodiment, the gtaB gene is selected fromthe group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 22; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 21; and (c) acomplementary strand of (a) or (b).

[0107] In another more preferred embodiment, the gtaB gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 22, or afragment thereof having UDP-glucose pyrophosphorylase activity.

[0108] In the methods of the present invention, theUDP-N-acetylglucosamine pyrophosphorylase gene may be a hasD or gcaDgene; or homologs thereof.

[0109] In a preferred embodiment, the hasD gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 75%, about 80%, about85%, about 90%, or about 95% identity to SEQ ID NO: 45; (b) a nucleicacid sequence which hybridizes under low, medium, or high stringencyconditions with SEQ ID NO: 44; and (c) a complementary strand of (a) or(b).

[0110] In another more preferred embodiment, the hasD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 45, or afragment thereof having UDP-N-acetylglucosamine pyrophosphorylaseactivity.

[0111] In another preferred embodiment, the gcaD gene is selected fromthe group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 30; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 29; and (c) acomplementary strand of (a) or (b).

[0112] In another more preferred embodiment, the gcaD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 30, or afragment thereof having UDP-N-acetylglucosamine pyrophosphorylaseactivity.

[0113] In the methods of the present invention, the glucose-6-phosphateisomerase gene may be a hasE or homolog thereof.

[0114] In a preferred embodiment, the hasE gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 101; (b)a nucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 100; and (c) a complementarystrand of (a) or (b).

[0115] In another more preferred embodiment, the hasE gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 101, or afragment thereof having glucose-6-phosphate isomerase activity.

[0116] In the methods of the present invention, the hyaluronan synthasegene and the one or more genes encoding a precursor sugar are under thecontrol of the same promoter. Alternatively, the one or more genesencoding a precursor sugar are under the control of the same promoterbut a different promoter driving the hyaluronan synthase gene. A furtheralternative is that the hyaluronan synthase gene and each of the genesencoding a precursor sugar are under the control of different promoters.In a preferred embodiment, the hyaluronan synthase gene and the one ormore genes encoding a precursor sugar are under the control of the samepromoter.

[0117] The present invention also relates to a nucleic acid constructcomprising an isolated nucleic acid sequence encoding a hyaluronansynthase operon comprising a hyaluronan synthase gene and a UDP-glucose6-dehydrogenase gene, and optionally one or more genes selected from thegroup consisting of a UDP-glucose pyrophosphorylase gene,UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphateisomerase gene. A nucleic acid sequence encoding most of the hyaluronansynthase operon of Streptococcus equisimilis is found in SEQ ID NO: 108.This sequence contains the hasB (SEQ ID NO: 40) and hasC (SEQ ID nO: 42)homologs of the Bacillus subtilis tuaD gene (SEQ ID NO: 11) and gtaBgene (SEQ ID NO: 21), respectively, as is the case for Streptococcuspyogenes, as well as a homolog of the gcaD gene (SEQ ID NO: 29), whichhas been designated hasD (SEQ ID NO: 44). The Bacillus subtilis gcaDencodes UDP-N-acetylglucosamine pyrophosphorylase, which is involved inthe synthesis of N-acetyl-glucosamine, one of the two sugars ofhyaluronan. The Streptococcus equisimilis homolog of gcaD, hasD, isarranged by Streptococcus equisimilis on the hyaluronan synthase operon.The nucleic aci sequence also contains a portion of the hasA gene (thelast 1156 bp of SEQ ID NO: 1).

[0118] In some cases the host cell will have a recombinant constructwith a heterologous promoter region operably linked to a nucleic acidsequence encoding a gene directing a step in the synthesis pathway of aprecursor sugar of hyaluronan, which may be in concert with theexpression of hyaluronan synthase from a recombinant construct. Thehyaluronan synthase may be expressed from the same or a differentpromoter region than the nucleic acid sequence encoding an enzymeinvolved in the biosynthesis of the precursor. In another preferredembodiment, the host cell may have a recombinant construct with apromoter region operably linked to a different nucleic acid sequenceencoding a second gene involved in the synthesis of a precursor sugar ofhyaluronan.

[0119] The nucleic acid sequence encoding the enzymes involved in thebiosynthesis of the precursor sugar(s) may be expressed from the same ora different promoter as the nucleic acid sequence encoding thehyaluronan synthase. In the former sense, “artificial operons” areconstructed, which may mimic the operon of Streptococcus equisimilis inhaving each hasA, hasB, hasC and hasD, or homologs thereof, or,alternatively, may utilize less than the full complement present in theStreptococcus equisimilis operon. The artificial operons” may alsocomprise a glucose-6-phosphate isomerase gene (hasE) as well as one ormore genes selected from the group consisting of a hexokinase gene,phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyltransferase gene. In the artificial operon, at least one of the elementsis heterologous to one other of the elements, such as the promoterregion being heterologous to the encoding sequences.

[0120] In a preferred embodiment, the nucleic acid construct compriseshasA, tuaD, and gtaB. In another preferred embodiment, the nucleic acidconstruct comprises hasA, tuaD, gtaB, and gcaD. In another preferredembodiment, the nucleic acid construct comprises hasA and tuaD. Inanother preferred embodiment, the nucleic acid construct comprises hasA.In another preferred embodiment, the nucleic acid construct compriseshasA, tuaD, gtaB, gcaD, and hasE. In another preferred embodiment, thenucleic acid construct comprises hasA, hasB, hasC, and hasD. In anotherpreferred embodiment, the nucleic acid construct comprises hasA, hasB,hasC, hasD, and hasE. Based on the above preferred embodiments, thegenes noted can be replaced with homologs thereof.

[0121] In the methods of the present invention, the nucleic acidconstructs comprise a hyaluronan synthase encoding sequence operablylinked to a promoter sequence foreign to the hyaluronan synthaseencoding sequence. The promoter sequence may be, for example, a singlepromoter or a tandem promoter.

[0122] “Promoter” is defined herein as a nucleic acid sequence involvedin the binding of RNA polymerase to initiate transcription of a gene.“Tandem promoter” is defined herein as two or more promoter sequenceseach of which is operably linked to a coding sequence and mediates thetranscription of the coding sequence into mRNA. “Operably linked” isdefined herein as a configuration in which a control sequence, e.g., apromoter sequence, is appropriately placed at a position relative to acoding sequence such that the control sequence directs the production ofa polypeptide encoded by the coding sequence. As noted earlier, a“coding sequence” is defined herein as a nucleic acid sequence which istranscribed into mRNA and translated into a polypeptide when placedunder the control of the appropriate control sequences. The boundariesof the coding sequence are generally determined by a ribosome bindingsite located just upstream of the open reading frame at the 5′ end ofthe mRNA and a transcription terminator sequence located just downstreamof the open reading frame at the 3′ end of the mRNA. A coding sequencecan include, but is not limited to, genomic DNA, cDNA, semisynthetic,synthetic, and recombinant nucleic acid sequences.

[0123] In a preferred embodiment, the promoter sequences may be obtainedfrom a bacterial source. In a more preferred embodiment, the promotersequences may be obtained from a gram positive bacterium such as aBacillus strain, e.g., Bacillus agaradherens, Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans orStreptomyces murinus; or from a gram negative bacterium, e.g., E. colior Pseudomonas sp.

[0124] Examples of suitable promoters for directing the transcription ofa nucleic acid sequence in the methods of the present invention are thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus lentus or Bacillus clausii alkalineprotease gene (aprH), Bacillus licheniformis alkaline protease gene(subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformisalpha-amylase gene (amyL), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionisCrylllA gene (crylllA) or portions thereof, prokaryotic beta-lactamasegene (Villa-Kamaroff et al., 1978, Proceedings of the National Academyof Sciences USA 75:3727-3731). Other examples are the promoter of thespol bacterial phage promoter and the tac promoter (DeBoer et al., 1983,Proceedings of the National Academy of Sciences USA 80:21-25). Furtherpromoters are described in “Useful proteins from recombinant bacteria”in Scientific American, 1980, 242:74-94; and in Sambrook, Fritsch, andManiatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.

[0125] The promoter may also be a “consensus” promoter having thesequence TTGACA for the “−35” region and TATAAT for the “−10” region.The consensus promoter may be obtained from any promoter which canfunction in a Bacillus host cell. The construction of a “consensus”promoter may be accomplished by site-directed mutagenesis to create apromoter which conforms more perfectly to the established consensussequences for the “−10” and “−35” regions of the vegetative “sigmaA-type” promoters for Bacillus subtilis (Voskuil et al., 1995, MolecularMicrobiology 17: 271-279).

[0126] In a preferred embodiment, the “consensus” promoter is obtainedfrom a promoter obtained from the E. coli lac operon, Streptomycescoelicolor agarase gene (dagA), Bacillus clausii or Bacillus lentusalkaline protease gene (aprH), Bacillus licheniformis alkaline proteasegene (subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene(sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionisCrylllA gene (crylllA) or portions thereof, or prokaryoticbeta-lactamase gene spo1 bacterial phage promoter. In a more preferredembodiment, the “consensus” promoter is obtained from Bacillusamyloliquefaciens alpha-amylase gene (amyQ).

[0127] Widner, et al., U.S. Pat. Nos. 6,255,076 and 5,955,310, describetandem promoters and constructs and methods for use in expression inBacillus cells, including the short consensus amyQ promoter (also calledscBAN). The use of the crylllA stabilizer sequence, and constructs usingthe sequence, for improved production in Bacillus are also describedtherein.

[0128] Each promoter sequence of the tandem promoter may be any nucleicacid sequence which shows transcriptional activity in the Bacillus cellof choice including a mutant, truncated, and hybrid promoter, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the Bacillus cell. Each promotersequence may be native or foreign to the nucleic acid sequence encodingthe polypeptide and native or foreign to the Bacillus cell. The promotersequences may be the same promoter sequence or different promotersequences.

[0129] The two or more promoter sequences of the tandem promoter maysimultaneously promote the transcription of the nucleic acid sequence.Alternatively, one or more of the promoter sequences of the tandempromoter may promote the transcription of the nucleic acid sequence atdifferent stages of growth of the Bacillus cell.

[0130] In a preferred embodiment, the tandem promoter contains at leastthe amyQ promoter of the Bacillus amyloliquefaciens alpha-amylase gene.In another preferred embodiment, the tandem promoter contains at least a“consensus” promoter having the sequence TTGACA for the “−35” region andTATAAT for the “−10” region. In another preferred embodiment, the tandempromoter contains at least the amyL promoter of the Bacilluslicheniformis alpha-amylase gene. In another preferred embodiment, thetandem promoter contains at least the crylllA promoter or portionsthereof (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107).

[0131] In a more preferred embodiment, the tandem promoter contains atleast the amyL promoter and the crylllA promoter. In another morepreferred embodiment, the tandem promoter contains at least the amyQpromoter and the crylllA promoter. In another more preferred embodiment,the tandem promoter contains at least a “consensus” promoter having thesequence TTGACA for the “−35” region and TATAAT for the “−10” region andthe crylllA promoter. In another more preferred embodiment, the tandempromoter contains at least two copies of the amyL promoter. In anothermore preferred embodiment, the tandem promoter contains at least twocopies of the amyQ promoter. In another more preferred embodiment, thetandem promoter contains at least two copies of a “consensus” promoterhaving the sequence TTGACA for the “−35” region and TATAAT for the “−10”region. In another more preferred embodiment, the tandem promotercontains at least two copies of the crylllA promoter.

[0132] “An mRNA processing/stabilizing sequence” is defined herein as asequence located downstream of one or more promoter sequences andupstream of a coding sequence to which each of the one or more promotersequences are operably linked such that all mRNAs synthesized from eachpromoter sequence may be processed to generate mRNA transcripts with astabilizer sequence at the 5′ end of the transcripts. The presence ofsuch a stabilizer sequence at the 5′ end of the mRNA transcriptsincreases their half-life (Agaisse and Lereclus, 1994, supra, Hue etal., 1995, Journal of Bacteriology 177: 3465-3471). The mRNAprocessing/stabilizing sequence is complementary to the 3′ extremity ofa bacterial 16S ribosomal RNA. In a preferred embodiment, the mRNAprocessing/stabilizing sequence generates essentially single-sizetranscripts with a stabilizing sequence at the 5′ end of thetranscripts. The mRNA processing/stabilizing sequence is preferably one,which is complementary to the 3′ extremity of a bacterial 16S ribosomalRNA. See, U.S. Pat. Nos. 6,255,076 and 5,955,310.

[0133] In a more preferred embodiment, the mRNA processing/stabilizingsequence is the Bacillus thuringiensis crylllA mRNAprocessing/stabilizing sequence disclosed in WO 94/25612 and Agaisse andLereclus, 1994, supra, or portions thereof which retain the mRNAprocessing/stabilizing function. In another more preferred embodiment,the mRNA processing/stabilizing sequence is the Bacillus subtilis SP82mRNA processing/stabilizing sequence disclosed in Hue et al., 1995,supra, or portions thereof which retain the mRNA processing/stabilizingfunction.

[0134] When the crylllA promoter and its mRNA processing/stabilizingsequence are employed in the methods of the present invention, a DNAfragment containing the sequence disclosed in WO 94/25612 and Agaisseand Lereclus, 1994, supra, or portions thereof which retain the promoterand mRNA processing/stabilizing functions, may be used. Furthermore, DNAfragments containing only the crylllA promoter or only the crylllA mRNAprocessing/stabilizing sequence may be prepared using methods well knownin the art to construct various tandem promoter and mRNAprocessing/stabilizing sequence combinations. In this embodiment, thecrylllA promoter and its mRNA processing/stabilizing sequence arepreferably placed downstream of the other promoter sequence(s)constituting the tandem promoter and upstream of the coding sequence ofthe gene of interest.

[0135] The isolated nucleic acid sequence encoding the desired enzyme(s)involved in hyaluronic acid production may then be further manipulatedto improve expression of the nucleic acid sequence. Expression will beunderstood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. The techniques for modifying nucleic acidsequences utilizing cloning methods are well known in the art.

[0136] A nucleic acid construct comprising a nucleic acid sequenceencoding an enzyme may be operably linked to one or more controlsequences capable of directing the expression of the coding sequence ina Bacillus cell under conditions compatible with the control sequences.

[0137] The term “control sequences” is defined herein to include allcomponents which are necessary or advantageous for expression of thecoding sequence of a nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the enzyme. Inaddition to promoter sequences described above, such control sequencesinclude, but are not limited to, a leader, a signal sequence, and atranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding an enzyme.

[0138] The control sequence may also be a suitable transcriptionterminator sequence, a sequence recognized by a Bacillus cell toterminate transcription. The terminator sequence is operably linked tothe 3′ terminus of the nucleic acid sequence encoding the enzyme or thelast enzyme of an operon. Any terminator which is functional in theBacillus cell of choice may be used in the present invention.

[0139] The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by theBacillus cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the enzyme. Any leader sequencewhich is functional in the Bacillus cell of choice may be used in thepresent invention.

[0140] The control sequence may also be a signal peptide coding region,which codes for an amino acid sequence linked to the amino terminus of apolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway. The signal peptide coding region may be native to thepolypeptide or may be obtained from foreign sources. The 5′ end of thecoding sequence of the nucleic acid sequence may inherently contain asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region which encodes the secretedpolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding region which is foreign to that portionof the coding sequence which encodes the secreted polypeptide. Theforeign signal peptide coding region may be required where the codingsequence does not normally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to obtainenhanced secretion of the polypeptide relative to the natural signalpeptide coding region normally associated with the coding sequence. Thesignal peptide coding region may be obtained from an amylase or aprotease gene from a Bacillus species. However, any signal peptidecoding region capable of directing the expressed polypeptide into thesecretory pathway of a Bacillus cell of choice may be used in thepresent invention.

[0141] An effective signal peptide coding region for Bacillus cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), and theBacillus subtilis prsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0142] The control sequence may also be a propeptide coding region thatcodes for an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE) and Bacillussubtilis neutral protease (nprT).

[0143] Where both signal peptide and propeptide regions are present atthe amino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

[0144] It may also be desirable to add regulatory sequences which allowthe regulation of the expression of the polypeptide relative to thegrowth of the host cell. Examples of regulatory systems are those whichcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems.

[0145] Expression Vectors

[0146] In the methods of the present invention, a recombinant expressionvector comprising a nucleic acid sequence, a promoter, andtranscriptional and translational stop signals may be used for therecombinant production of an enzyme involved in hyaluronic acidproduction. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide or enzyme at such sites. Alternatively, the nucleic acidsequence may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression,and possibly secretion.

[0147] The recombinant expression vector may be any vector which can beconveniently subjected to recombinant DNA procedures and can bring aboutthe expression of the nucleic acid sequence. The choice of the vectorwill typically depend on the compatibility of the vector with theBacillus cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the Bacillus cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The vector system may be a single vector or plasmidor two or more vectors or plasmids which together contain the total DNAto be introduced into the genome of the Bacillus cell, or a transposonmay be used.

[0148] The vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the Bacillus hostcell's genome or autonomous replication of the vector in the cellindependent of the genome.

[0149] For integration into the host cell genome, the vector may rely onthe nucleic acid sequence encoding the polypeptide or any other elementof the vector for integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the Bacillus cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the Bacillus cell genome at a precise location in the chromosome.To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the Bacillus cell. Furthermore, the integrational elements maybe non-encoding or encoding nucleic acid sequences. On the other hand,the vector may be integrated into the genome of the host cell bynon-homologous recombination.

[0150] For autonomous replication, the vector may further comprise anorigin of replication enabling the vector to replicate autonomously inthe Bacillus cell in question. Examples of bacterial origins ofreplication are the origins of replication of plasmids pUB110, pE194,pTA1060, and pAMβ1 permitting replication in Bacillus. The origin ofreplication may be one having a mutation to make its functiontemperature-sensitive in the Bacillus cell (see, e.g., Ehrlich, 1978,Proceedings of the National Academy of Sciences USA 75:1433).

[0151] The vectors preferably contain one or more selectable markerswhich permit easy selection of transformed cells. A selectable marker isa gene the product of which provides for biocide resistance, resistanceto heavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Furthermore, selection may be accomplished byco-transformation, e.g., as described in WO 91/09129, where theselectable marker is on a separate vector.

[0152] More than one copy of a nucleic acid sequence may be insertedinto the host cell to increase production of the gene product. Anincrease in the copy number of the nucleic acid sequence can be obtainedby integrating at least one additional copy of the sequence into thehost cell genome or by including an amplifiable selectable marker genewith the nucleic acid sequence where cells containing amplified copiesof the selectable marker gene, and thereby additional copies of thenucleic acid sequence, can be selected for by cultivating the cells inthe presence of the appropriate selectable agent. A convenient methodfor achieving amplification of genomic DNA sequences is described in WO94/14968.

[0153] The procedures used to ligate the elements described above toconstruct the recombinant expression vectors are well known to oneskilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0154] Production

[0155] In the methods of the present invention, the Bacillus host cellsare cultivated in a nutrient medium suitable for production of thehyaluronic acid using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the enzymes involved inhyaluronic acid synthesis to be expressed and the hyaluronic acid to beisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted hyaluronic acid can be recovered directly fromthe medium.

[0156] The resulting hyaluronic acid may be isolated by methods known inthe art. For example, the hyaluronic acid may be isolated from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation. The isolated hyaluronic acid may then be furtherpurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,N.Y., 1989).

[0157] In the methods of the present invention, the Bacillus host cellsproduce greater than about 4 g, preferably greater than about 6 g, morepreferably greater than about 8 g, even more preferably greater thanabout 10 g, and most preferably greater than about 12 g of hyaluronicacid per liter.

[0158] Deletions/Disruptions

[0159] Gene deletion or replacement techniques may be used for thecomplete removal of a selectable marker gene or other undesirable gene.In such methods, the deletion of the selectable marker gene may beaccomplished by homologous recombination using a plasmid that has beenconstructed to contiguously contain the 5′ and 3′ regions flanking theselectable marker gene. The contiguous 5′ and 3′ regions may beintroduced into a Bacillus cell on a temperature-sensitive plasmid,e.g., pE194, in association with a second selectable marker at apermissive temperature to allow the plasmid to become established in thecell. The cell is then shifted to a non-permissive temperature to selectfor cells that have the plasmid integrated into the chromosome at one ofthe homologous flanking regions. Selection for integration of theplasmid is effected by selection for the second selectable marker. Afterintegration, a recombination event at the second homologous flankingregion is stimulated by shifting the cells to the permissive temperaturefor several generations without selection. The cells are plated toobtain single colonies and the colonies are examined for loss of bothselectable markers (see, for example, Perego, 1993, In A. L. Sonneshein,J. A. Hoch, and R. Losick, editors, Bacillus subtilis and OtherGram-Positive Bacteria, Chapter 42, American Society of Microbiology,Washington, D.C., 1993).

[0160] A selectable marker gene may also be removed by homologousrecombination by introducing into the mutant cell a nucleic acidfragment comprising 5′ and 3′ regions of the defective gene, but lackingthe selectable marker gene, followed by selecting on thecounter-selection medium. By homologous recombination, the defectivegene containing the selectable marker gene is replaced with the nucleicacid fragment lacking the selectable marker gene. Other methods known inthe art may also be used.

[0161] U.S. Pat. No. 5,891,701 discloses techniques for deleting severalgenes including spollAC, aprE, nprE, and amyE.

[0162] Other undesirable biological compounds may also be removed by theabove described methods such as the red pigment synthesized by cypX(accession no. BG12580) and/or yvmC (accession no. BG14121).

[0163] In a preferred embodiment, the Bacillus host cell is unmarkedwith any heterologous or exogenous selectable markers. In anotherpreferred embodiment, the Bacillus host cell does not produce any redpigment synthesized by cypX and yvmC.

[0164] Isolated Nucleic Acid Sequences Encoding Polypeptides HavingUDP-Glucose 6-Dehydrogenase Activity, UDP-Glucose PyrophosphorylaseActivity, or UDP-N-Acetylglucosamine Pyrophosphorylase Activity

[0165] The term “UDP-glucose 6-dehydrogenase activity” is defined hereinas a UDP glucose:NAD⁺6-oxidoreductase activity which catalyzes theconversion of UDP-glucose in the presence of 2AND⁺and water toUDP-glucuronate and 2NADH. For purposes of the present inventionUDP-glucose 6-dehydrogenase activity is determined according to theprocedure described by Jaenicke and Rudolph, 1986, Biochemistry 25:7283-7287. One unit of UDP-glucose 6-dehydrogenase activity is definedas 1.0 μmole of UDP-glucuronate produced per minute at 25° C., pH 7.

[0166] The term “UDP-glucose pyrophosphorylase activity” is definedherein as a UTP:□-D-glucose-1-phosphate uridylyltransferase activitywhich catalyzes the conversion of glucose-1-phosphate in the presence ofUTP to diphosphate and UDP-glucose. For purposes of the presentinvention UDP-glucose pyrophosphorylase activity activity is determinedaccording to the procedure described by Kamogawa et al., 1965, J.Biochem. (Tokyo) 57: 758-765 or Hansen et al., 1966, Method Enzymol. 8:248-253. One unit of UDP-glucose pyrophosphorylase activity is definedas 1.0 μmole of UDP-glucose produced per minute at 25° C., pH 7.

[0167] The term “UDP-N-acetylglucosamine pyrophosphorylase activity” isdefined herein as a UTP:N-acetyl-alpha-D-glucoamine-1-phosphateuridyltransferase activity which catalyzes the conversion ofN-acetyl-alpha-D-glucosamine-1-phosphate in the presence of UTP todiphosphate and UDP-N-acetyl-alpha-D-glucoamine. For purposes of thepresent invention, UDP-N-acetylglucosamine pyrophosphorylase activity isdetermined according to the procedure described by Mangin-Lecreuix etal., 1994, J. Bacteriology 176: 5788-5795. One unit ofUDP-N-acetylglucosamine pyrophosphorylase activity is defined as 1.0μmole of UDP-N-acetyl-alpha-D-glucoamine produced per minute at 25° C.,pH 7.

[0168] The term “isolated nucleic acid sequence” as used herein refersto a nucleic acid sequence which is essentially free of other nucleicacid sequences, e.g., at least about 20% pure, preferably at least about40% pure, more preferably at least about 60% pure, even more preferablyat least about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

[0169] In a first embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having an amino acidsequence which has a degree of identity to SEQ ID NO: 41 of at leastabout 75%, preferably at least about 80%, more preferably at least about85%, even more preferably at least about 90%, most preferably at leastabout 95%, and even most preferably at least about 97%, which haveUDP-glucose 6-dehydrogenase activity (hereinafter “homologouspolypeptides”). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from SEQ ID NO: 41.

[0170] In another first embodiment, the present invention relates toisolated nucleic acid sequences encoding polypeptides having an aminoacid sequence which has a degree of identity to SEQ ID NO: 43 of atleast about 90%, preferably at least about 95%, and more preferably atleast about 97%, which have UDP-glucose pyrophosphorylase activity(hereinafter “homologous polypeptides”). In a preferred embodiment, thehomologous polypeptides have an amino acid sequence which differs byfive amino acids, preferably by four amino acids, more preferably bythree amino acids, even more preferably by two amino acids, and mostpreferably by one amino acid from SEQ ID NO: 43.

[0171] In another first embodiment, the present invention relates toisolated nucleic acid sequences encoding polypeptides having an aminoacid sequence which has a degree of identity to SEQ ID NO: 45 of atleast about 75%, preferably at least about 80%, more preferably at leastabout 85%, even more preferably at least about 90%, most preferably atleast about 95%, and even most preferably at least about 97%, which haveUDP-N-acetylglucosamine pyrophosphorylase activity (hereinafter“homologous polypeptides”). In a preferred embodiment, the homologouspolypeptides have an amino acid sequence which differs by five aminoacids, preferably by four amino acids, more preferably by three aminoacids, even more preferably by two amino acids, and most preferably byone amino acid from SEQ ID NO: 45.

[0172] For purposes of the present invention, the degree of identitybetween two amino acid sequences is determined by the Clustal method(Higgins, 1989, CABIOS 5: 151-153) using the Vector NTI AlignX softwarepackage (Informax Inc., Bethesda, Md.) with the following defaults:pairwise alignment, gap opening penalty of 10, gap extension penalty of0.1, and score matrix: blosum62mt2.

[0173] Preferably, the nucleic acid sequences of the present inventionencode polypeptides that comprise the amino acid sequence of SEQ ID NO:41, SEQ ID NO: 43, or SEQ ID NO: 45; or an allelic variant thereof; or afragment thereof that has UDP-glucose 6-dehydrogenase, UDP-glucosepyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylaseactivity, respectively. In a more preferred embodiment, the nucleic acidsequence of the present invention encodes a polypeptide that comprisesthe amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO:45. In another preferred embodiment, the nucleic acid sequence of thepresent invention encodes a polypeptide that consists of the amino acidsequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or anallelic variant thereof; or a fragment thereof, wherein the polypeptidefragment has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase,or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively. Inanother preferred embodiment, the nucleic acid sequence of the presentinvention encodes a polypeptide that consists of the amino acid sequenceof SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.

[0174] The present invention also encompasses nucleic acid sequenceswhich encode a polypeptide having the amino acid sequence of SEQ ID NO:41, SEQ ID NO: 43, or SEQ ID NO: 45, which differ from SEQ ID NO: 40,SEQ ID NO: 42, or SEQ ID NO: 44 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44 which encode fragments of SEQID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, respectively, which haveUDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity, respectively.

[0175] A subsequence of SEQ ID NO: 40 is a nucleic acid sequenceencompassed by SEQ ID NO: 40 except that one or more nucleotides fromthe 5′ and/or 3′ end have been deleted. Preferably, a subsequencecontains at least 1020 nucleotides, more preferably at least 1080nucleotides, and most preferably at least 1140 nucleotides. A fragmentof SEQ ID NO: 41 is a polypeptide having one or more amino acids deletedfrom the amino and/or carboxy terminus of this amino acid sequence.Preferably, a fragment contains at least 340 amino acid residues, morepreferably at least 360 amino acid residues, and most preferably atleast 380 amino acid residues.

[0176] A subsequence of SEQ ID NO: 42 is a nucleic acid sequenceencompassed by SEQ ID NO: 42 except that one or more nucleotides fromthe 5′ and/or 3′ end have been deleted. Preferably, a subsequencecontains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides. A fragment ofSEQ ID NO: 43 is a polypeptide having one or more amino acids deletedfrom the amino and/or carboxy terminus of this amino acid sequence.Preferably, a fragment contains at least 255 amino acid residues, morepreferably at least 270 amino acid residues, and most preferably atleast 285 amino acid residues.

[0177] A subsequence of SEQ ID NO: 44 is a nucleic acid sequenceencompassed by SEQ ID NO: 44 except that one or more nucleotides fromthe 5′ and/or 3′ end have been deleted. Preferably, a subsequencecontains at least 1110 nucleotides, more preferably at least 1200nucleotides, and most preferably at least 1290 nucleotides. A fragmentof SEQ ID NO: 45 is a polypeptide having one or more amino acids deletedfrom the amino and/or carboxy terminus of this amino acid sequence.Preferably, a fragment contains at least 370 amino acid residues, morepreferably at least 400 amino acid residues, and most preferably atleast 430 amino acid residues.

[0178] An allelic variant denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. The allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

[0179] In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to SEQ ID NO: 40of at least about 75%, preferably at least about 80%, more preferably atleast about 85%, even more preferably at least about 90%, mostpreferably at least about 95%, and even most preferably at least about97%.

[0180] In another second embodiment, the present invention relates toisolated nucleic acid sequences which have a degree of homology to SEQID NO: 42 of at least about 90%, preferably at least about 95%, and morepreferably at least about 97%.

[0181] In another second embodiment, the present invention relates toisolated nucleic acid sequences which have a degree of homology to SEQID NO: 44 of at least about 75%, preferably at least about 80%, morepreferably at least about 85%, even more preferably at least about 90%,most preferably at least about 95%, and even most. preferably at leastabout 97%.

[0182] For purposes of the present invention, the degree of homologybetween two nucleic acid sequences is determined by the Vector NTIAlignX software package (Informax Inc., Bethesda, Md.) using thefollowing defaults: pairwise alignment, gap opening penalty of 15, gapextension penalty of 6.6, and score matrix: swgapdnamt.

[0183] In a third embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having UDP-glucose6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity, which hybridizeunder very low stringency conditions, preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 40, SEQ IDNO: 42, or SEQ ID NO: 44, (ii) the cDNA sequence contained in SEQ ID NO:40, SEQ ID NO: 42, or SEQ ID NO: 44, or (iii) a complementary strand of(i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). The subsequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44may be at least 100 nucleotides or preferably at least 200 nucleotides.Moreover, the respective subsequence may encode a polypeptide fragmentwhich has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity.

[0184] The nucleic acid sequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQID NO: 44, or subsequences thereof, as well as the amino acid sequenceof SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, or a fragmentthereof, may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosaminepyrophosphorylase activity, respectively, from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicor cDNA of the genus or species of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, preferably at least 25, and morepreferably at least 35 nucleotides in length. Longer probes can also beused. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

[0185] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having UDP-glucose6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity. Genomic or other DNAfrom such other organisms may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA which is homologous with SEQ ID NO: 40, SEQ ID NO: 42, orSEQ ID NO: 44, or a subsequence thereof, the carrier material is used ina Southern blot. For purposes of the present invention, hybridizationindicates that the nucleic acid sequence hybridizes to a labeled nucleicacid probe corresponding to the nucleic acid sequence shown in SEQ IDNO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

[0186] In a preferred embodiment, the nucleic acid probe is a nucleicacid sequence which encodes the polypeptide of SEQ ID NO: 41, SEQ ID NO:43, or SEQ ID NO: 45; or a subsequence thereof. In another preferredembodiment, the nucleic acid probe is SEQ ID NO: 40, SEQ ID NO: 42, orSEQ ID NO: 44. In another preferred embodiment, the nucleic acid probeis the nucleic acid sequence contained in plasmid pMRT106 which iscontained in Escherichia coli NRRL B-30536, wherein the nucleic acidsequence encodes polypeptides having UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosaminepyrophosphorylase activity.

[0187] For long probes of at least 100 nucleotides in length, very lowto very high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

[0188] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2 x SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0189] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridization, and washing post-hybridization at 5° C.to 10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

[0190] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6X SCCplus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6X SSCat 5° C. to 10° C. below the calculated T_(m).

[0191] In a fourth embodiment, the present invention relates to isolatednucleic acid sequences which encode variants of the polypeptide havingan amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45comprising a substitution, deletion, and/or insertion of one or moreamino acids.

[0192] The amino acid sequences of the variant polypeptides may differfrom the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ IDNO: 45, by an insertion or deletion of one or more amino acid residuesand/or the substitution of one or more amino acid residues by differentamino acid residues. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

[0193] Examples of conservative substitutions are within the group ofbasic amino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, N.Y. The mostcommonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

[0194] Modification of a nucleic acid sequence of the present inventionmay be necessary for the synthesis of polypeptides substantially similarto the polypeptide. The term “substantially similar” to the polypeptiderefers to non-naturally occurring forms of the polypeptide. Thesepolypeptides may differ in some engineered way from the polypeptideisolated from its native source, e.g., variants that differ in specificactivity, thermostability, pH optimum, or the like. The variant sequencemay be constructed on the basis of the nucleic acid sequence presentedas the polypeptide encoding part of SEQ ID NO: 40, SEQ ID NO: 42, or SEQID NO: 44, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich corresponds to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

[0195] It will be apparent to those skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidresidues essential to the activity of the polypeptide encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for enzyme activity to identifyamino acid residues that are critical to the activity of the molecule.Sites of substrate-enzyme interaction can also be determined by analysisof the three-dimensional structure as determined by such techniques asnuclear magnetic resonance analysis, crystallography or photoaffinitylabelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smithet al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver etal., 1992, FEBS Letters 309: 59-64).

[0196] The polypeptides encoded by the isolated nucleic acid sequencesof the present invention have at least 20%, preferably at least 40%,more preferably at least 60%, even more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 100% of theUDP-glucose 6-dehydrogenase activity of the polypeptide of SEQ ID NO:41, the UDP-glucose pyrophosphorylase activity of the polypeptide of SEQID NO: 43, or the UDP-N-acetylglucosamine pyrophosphorylase activity ofthe polypeptide of SEQ ID NO: 45.

[0197] The nucleic acid sequences of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by the nucleic acidsequence is produced by the source or by a cell in which the nucleicacid sequence from the source has been inserted. In a preferredembodiment, the polypeptide encoded by a nucleic acid sequence of thepresent invention is secreted extracellularly.

[0198] The nucleic acid sequences may be obtained from a bacterialsource. For example, these polypeptides may be obtained from a grampositive bacterium such as a Bacillus strain, e.g., Bacillusagaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, orBacillus thuringiensis; or a Streptomyces strain, e.g., Streptomyceslividans or Streptomyces murinus; or from a gram negative bacterium,e.g., E. coli or Pseudomonas sp.

[0199] In a preferred embodiment, the nucleic acid sequences areobtained from a Streptococcus or Pastuerella strain.

[0200] In a more preferred embodiment, the nucleic acid sequences areobtained from a Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, or Streptococcus equi subs. zooepidemicus strain,or a Pasteurella multocida strain.

[0201] In a most preferred embodiment, the nucleic acid sequences areobtained from Streptococcus equisimilis, e.g., the nucleic acid sequenceset forth in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In anothermost preferred embodiment, the nucleic acid sequence is the sequencecontained in plasmid pMRT106 which is contained in Escherichia coli NRRLB-30536. In further most preferred embodiment, the nucleic acid sequenceis SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0202] Strains of these species are readily accessible to the public ina number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Pat. Culture Collection, Northern RegionalResearch Center (NRRL).

[0203] Furthermore, such nucleic acid sequences may be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) using the above-mentionedprobes. Techniques for isolating microorganisms from natural habitatsare well known in the art. The nucleic acid sequence may then be derivedby similarly screening a genomic or cDNA library of anothermicroorganism. Once a nucleic acid sequence encoding a polypeptide hasbeen detected with the probe(s), the sequence may be isolated or clonedby utilizing techniques which are known to those of ordinary skill inthe art (see, e.g., Sambrook et a., 1989, supra).

[0204] The present invention also relates to mutant nucleic acidsequences comprising at least one mutation in the polypeptide codingsequence of SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, in whichthe mutant nucleic acid sequence encodes a polypeptide which consists ofSEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45, respectively.

[0205] The techniques used to isolate or clone a nucleic acid sequenceencoding a polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequences of the present invention from suchgenomic DNA can be effected, e.g., by using the well known polymerasechain reaction (PCR) or antibody screening of expression libraries todetect cloned DNA fragments with shared structural features. See, e.g.,Innis et al., 1990, PCR: A Guide to Methods and Application, AcademicPress, N.Y. Other nucleic acid amplification procedures such as ligasechain reaction (LCR), ligated activated transcription (LAT) and nucleicacid sequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Streptococcus, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

[0206] The present invention also relates to nucleic acid constructscomprising a nucleic acid sequence of the present invention operablylinked to one or more control sequences which direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

[0207] The present invention also relates to recombinant expressionvectors comprising a nucleic acid sequence of the present invention, apromoter, and transcriptional and translational stop signals.

[0208] The present invention also relates to recombinant host cells,comprising a nucleic acid sequence of the invention, which areadvantageously used in the recombinant production of the polypeptides.

[0209] The present invention also relates to methods for producing apolypeptide having UDP-N-acetylglucosamine pyrophosphorylase activitycomprising (a) cultivating a host cell under conditions suitable forproduction of the polypeptide; and (b) recovering the polypeptide.

[0210] In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

[0211] The polypeptides may be detected using methods known in the artthat are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide as describedherein.

[0212] The resulting polypeptide may be recovered by methods known inthe art. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

[0213] The polypeptides may be purified by a variety of procedures knownin the art including, but not limited to, chromatography (e.g., ionexchange, affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, N.Y., 1989).

[0214] The present invention further relates to the isolatedpolypeptides having UDP-glucose 6-dehydrogenase, UDP-glucosepyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylase activityencoded by the nucleic acid sequences described above.

[0215] The present invention is further described by the followingexamples which should not be construed as limiting the scope of theinvention.

EXAMPLES Primers and Oligos

[0216] All primers and oligos were purchased (MWG Biotech Inc., HighPoint, N.C.)

Example 1: PCR amplification and cloning of the Streptococcusequisimilis hasA gene and the Bacillus subtilis tuaD, gtaB, and gcaDgenes

[0217] The Streptococcus equisimilis hyaluronan synthase gene (hasA,accession number AF023876, SEQ ID NOs: 1 [DNA sequence] and 2 [deducedamino acid sequence]) was PCR amplified from plasmid pKKseD (Weigel,1997, Journal of Biological Chemistry 272: 32539-32546) using primers 1and 2: Primer 1: (SEQ ID NO: 3)5′-GAGCTCTATAAAAATGAGGAGGGAACCGAATGAGAACATTAAAAAAC CT-3′ Primer 2: (SEQID NO: 4) 5′-GTTAACGAATTCAGCTATGTAGGTACCTTATAATAATTTTTTACGTG T-3′

[0218] PCR amplifications were conducted in triplicate in 50 μlreactions composed of 1 ng of pKKseD DNA, 0.4 μM each of primers 1 and2, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1X PCR Buffer II (AppliedBiosystems, Inc., Foster City, Calif.) with 2.5 mM MgCl₂, and 2.5 unitsof AmpliTaq Gold™ DNA polymerase (Applied Biosystems, Inc., Foster City,Calif.). The reactions were performed in a RoboCycler 40 thermacycler(Stratagene, Inc., La Jolla, Calif.) programmed for 1 cycle at 95° C.for 9 minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1minute, and 72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for5 minutes. The PCR product was visualized using a 0.8% agarose gel with44 mM Tris Base, 44 mM boric acid, 0.5 mM EDTA buffer (0.5X TBE). Theexpected fragment was approximately 1200 bp.

[0219] The 1200 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOCloning Kit (Stratagene, Inc., La Jolla, Calif.) and transformed into E.coli OneShot™ competent cells according to the manufacturers'instructions (Stratagene, Inc., La Jolla, Calif.). Transformants wereselected at 37° C. after 16 hours of growth on 2X yeast-tryptone (YT)agar plates supplemented with 100 μg of ampicillin per ml. Plasmid DNAfrom these transformants was purified using a QIAGEN robot (QIAGEN,Valencia, Calif.) according to the manufacturer's instructions and theDNA sequence of the inserts confirmed by DNA sequencing using M13 (−20)forward and M13 reverse primers (Invitrogen, Inc, Carlsbad, Calif.) andthe following internal primers. The plasmid harboring the 1200 bp PCRfragment was designated pCR2.1-sehasA (FIG. 3). Primer 3:5′-GTTGACGATGGAAGTGCTGA-3′ (SEQ ID NO: 5) Primer 4:5′-ATCCGTTACAGGTAATATCC-3′ (SEQ ID NO: 6) Primer 5:5′-TCCTTTTGTAGCCCTATGGA-3′ (SEQ ID NO: 7) Primer 6:5′-TCAGCACTTCCATCGTCAAC-3′ (SEQ ID NO: 8) Primer 7:5′-GGATATTACCTGTAACGGAT-3′ (SEQ ID NO: 9) Primer 8:5′-TCCATAGGGCTACAAAAGGA-3′ (SEQ ID NO: 10)

[0220] The Bacillus subtilis UDP-glucose-6-dehydrogenase gene (tuaD,accession number BG12691, SEQ ID NOs: 11 [DNA sequence] and 12 [deducedamino acid sequence]) was PCR amplified from Bacillus subtilis 168 (BGSC1A1, Bacillus Genetic Stock Center, Columbus, Ohio) using primers 9 and10: Primer 9: (SEQ ID NO: 13) 5′-GGTACCGACACTGCGACCATTATAAA-3′ Primer10: (SEQ ID NO: 14) 5′-GTTAACGAATTCCAGCTATGTATCTAGACAGCTTCAACCAAGTAACACT-3′

[0221] PCR amplifications were carried out in triplicate in 30 μlreactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA,0.3 μM each of primers 9 and 10, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C.for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycleseach at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized in a 0.8% agarose gel using 0.5X TBE buffer. The expectedfragment was approximately 1400 bp.

[0222] The 1400 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOCloning Kit and transformed into E. coli OneShot™ competent cellsaccording to the manufacturers' instructions. Plasmid DNA was purifiedusing a QIAGEN robot according to the manufacturer's instructions andthe DNA sequence of the inserts confirmed by DNA sequencing using M13(−20) forward and M13 reverse primers and the following internalprimers. The plasmid harboring the 1400 bp PCR fragment was designatedpCR2.1-tuaD (FIG. 4).

[0223] Primer 11: Primer 11: 5′-AGCATCTTAACGGCTACAAA-3′ (SEQ ID NO: 15)Primer 12: 5′-TGTGAGCGAGTCGGCGCAGA-3′ (SEQ ID NO: 16) Primer 13:5′-GGGCGCCCATGTAAAAGCAT-3′ (SEQ ID NO: 17) Primer 14:5′-TTTGTAGCCGTTAAGATGCT-3′ (SEQ ID NO: 18) Primer 15:5′-TCTGCGCCGACTCGCTCACA-3′ (SEQ ID NO: 19) Primer 16:5′-ATGCTTTTACATGGGCGCCC-3′ (SEQ ID NO: 20)

[0224] The Bacillus subtilis UTP-glucose-1-phosphate uridylyltransferasegene (gtaB, accession number BG10402, SEQ ID NOs: 21 [DNA sequence] and22 [deduced amino acid sequence]) was PCR amplified from Bacillussubtilis 168 using primers 17 and 18: Primer 17:5′-TCTAGATTTTTCGATCATAAGGAAGGT-3′ (SEQ ID NO: 23) Primer 18:5′-GTTAACGAATTCCAGCTATGTAGGATCCAATGTCCAATAGCCTTTTTGT-3′ (SEQ ID NO: 24)

[0225] PCR amplifications were carried out in triplicate in 30 μlreactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA,0.3 μM each of primers 17 and 18, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C.for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycleseach at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized in a 0.8% agarose-0.5X TBE gel. The expected fragment wasapproximately 900 bp.

[0226] The 900 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOcloning kit and transformed into E. coli OneShot™ competent cellsaccording to the manufacturer's instructions. Plasmid DNA was purifiedusing a QIAGEN robot according to the manufacturer's instructions andthe DNA sequence of the inserts confirmed by DNA sequencing using M13(−20) forward and M13 reverse primers and the following internalprimers. The plasmid harboring the 900 bp PCR fragment was designatedpCR2.1-gtaB (FIG. 5). Primer 19: 5′-AAAAAGGCTTCTAACCTGGC-3′ (SEQ ID NO:25) Primer 20: 5′-AAACCGCCTAAAGGCACAGC-3′ (SEQ ID NO: 26) Primer 21:5′-GCCAGGTTAGAAGCCTTTTT-3′ (SEQ ID NO: 27) Primer 22:5′-GCTGTGCCTTTAGGCGGTTT-3′ (SEQ ID NO: 28)

[0227] The Bacillus subtilis UDP-N-acetylglucosamine pyrophosphorylasegene (gcaD, accession number BG10113, SEQ ID NOs: 29 [DNA sequence] and30 [deduced amino acid sequence]) was PCR amplified from Bacillussubtilis 168 using primers 23 and 24: Primer 23:5′-GGATCCTTTCTATGGATAAAAGGGAT-3′ (SEQ ID NO: 31) Primer 24:5′-GTTAACAGGATTATTTTTTATGAATATTTTT-3′ (SEQ ID NO: 32)

[0228] PCR amplifications were carried out in triplicate in 30 μlreactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA,0.3 μM each of primers 23 and 24, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40programmed for 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C.for 1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycleseach at 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized in a 0.8% agarose-0.5X TBE gel. The expected fragment wasapproximately 1500 bp.

[0229] The 1500 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOcloning kit and transformed into E. coli OneShot™ competent cellsaccording to the manufacturer's instructions. Plasmid DNA was purifiedusing a QIAGEN robot according to the manufacturer's instructions andthe DNA sequence of the inserts confirmed by DNA sequencing using M13(−20) forward and M13 reverse primers and the following internalprimers. The plasmid harboring the 900 bp PCR fragment was designatedpCR2.1-gcaD (FIG. 6). Primer 25: 5′-CAGAGACGATGGAACAGATG-3′ (SEQ ID NO:33) Primer 26: 5′-GGAGTTAATGATAGAGTTGC-3′ (SEQ ID NO: 34) Primer 27:5′-GAAGATCGGGAATTTTGTAG-3′ (SEQ ID NO: 35) Primer 28:5′-CATCTGTTCCATCGTCTCTG-3′ (SEQ ID NO: 36) Primer 29:5′-GCAACTCTATCATTAACTCC-3′ (SEQ ID NO: 37) Primer 30:5′-CTACAAAATTCCCGATCTTC-3′ (SEQ ID NO: 38)

Example 2: Construction of the hasA/tuaD/gtaB operon

[0230] Plasmids pDG268Δneo-crylllAstab/Sav (U.S. Pat. No. 5,955,310) andpCR2.1-tuaD (Example 1, FIG. 4) were digested with Kpnl and Hpal. Thedigestions were resolved on a 0.8% agarose gel using 0.5X TBE buffer andthe larger vector fragment (approximately 7700 bp) frompDG268Δneo-crylllAstab/Sav and the smaller tuaD fragment (approximately1500 bp) from pCR2.1-tuaD were gel-purified using the QIAquick DNAExtraction kit according to the manufacturer's instructions (QIAGEN,Valencia, Calif.). The two purified fragments were ligated together withT4 DNA ligase according to the manufacturer's instructions (RocheApplied Science; Indianapolis, Ind.) and the ligation mix wastransformed into E. coli SURE competent cells (Stratagene, Inc., LaJolla, Calif.). Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml.

[0231] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby Kpnl plus Hpal digestion on a 0.8% agarose gel using 0.5X TBE buffer.The correct plasmid was identified by the presence of an approximately1500 bp Kpnl/Hpal tuaD fragment and was designated pHA1(FIG. 7).

[0232] Plasmids pHA1 and pCR2.1-gtaB (Example 1, FIG. 5) were digestedwith Xbal and Hpal. The digestions were resolved on a 0.8% agarose gelusing 0.5X TBE buffer and the larger vector fragment from pHA1(approximately 9200 bp) and the smaller gtaB fragment (approximately 900bp) from pCR2.1-gtaB were gel-purified from a 0.8% agarose-0.5X TBEbuffer gel using the QIAquick DNA Extraction Kit according to themanufacturer's instructions. These two purified fragments were ligatedtogether with T4 DNA ligase and the ligation mix was used to transformE. coli SURE competent cells. Transformants were selected on 2X YT agarplates supplemented with 100 μg of ampicillin per ml at 37° C.

[0233] Plasmids were purified from several transformants using a QIAGENrobot according to the manufacturer's instructions and analyzed by Xbalplus Hpal digestion. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The correct plasmid was identified by the presence of anapproximately 900 bp Xbal/Hpal gtaB fragment and was designated pHA2(FIG. 8).

[0234] Plasmids pHA2 and pCR2.1-sehasA (Example 1, FIG. 3) were digestedwith Sacl plus Kpnl. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The larger vector fragment (approximately 10000 bp) frompHA2 and the smaller hasA fragment (approximately 1300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5X TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml at 37° C. Plasmids werepurified from several transformants using a QIAGEN robot according tothe manufacturer's instructions and analyzed by Sacl plus Kpnldigestion. The digestions were resolved on a 0.8% agarose-0.5X TBEbuffer gel. The correct plasmid was identified by the presence of anapproximately 1300 bp Sacl/Kpnl hasA fragment and was designated pHA3(FIG. 9).

Example 3: Construction of the hasA/tuaD/gtaB/gcaD operon

[0235] Plasmids pHA2 (Example 2, FIG. 8) and pCR2.1-gcaD (Example 1,FIG. 6) were digested with BamHl and Hpal. The digestions were resolvedon a 0.8% agarose gel using 0.5X TBE buffer and the larger vectorfragment (approximately 10,000 bp) from pHA2 and the smaller gcaDfragment (approximately 1,400 bp) from pCR2.1-gcaD were gel-purifiedfrom a 0.8% agarose-0.5X TBE buffer gel using the QIAquick DNAExtraction Kit according to the manufacturer's instructions. These twopurified fragments were ligated together with T4 DNA ligase and theligation mix was used to transform E. coli SURE competent cells.Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml at 37° C.

[0236] Plasmids were purified from several transformants using a QIAGENrobot according to the manufacturer's instructions and analyzed by Xbalplus Hpal digestion. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The correct plasmid was identified by the presence of anapproximately 1400 bp BamHl/Hpal gcaD fragment and was designated pHA4(FIG. 10).

[0237] Plasmids pHA4 and pCR2.1-sehasA (Example 1, FIG. 3) were digestedwith Sacl and Kpnl. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The larger vector fragment (approximately 11,000 bp)from pHA4 and the smaller hasA fragment (approximately 1,300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5X TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml at 37° C. Plasmids werepurified from several transformants using a QIAGEN robot according tothe manufacturer's instructions and analyzed by Sacl plus Kpnldigestion. The digestions were resolved on a 0.8% agarose-0.5X TBEbuffer gel. The correct plasmid was identified by the presence of anapproximately 1,300 bp Sacl/Kpnl hasA fragment and was designated pHA5(FIG. 11).

Example 4: Construction of the hasA/tuaD/gcaD operon

[0238] Plasmids pHA1 (Example 2, FIG. 7) and pCR2.1-gcaD (Example 1,FIG. 6) were digested with BamHl and Hpal. The digestions were resolvedon a 0.8% agarose gel using 0.5X TBE buffer and the larger vectorfragment from pHA1 (approximately 9,200 bp) and the smaller gcaDfragment (approximately 1400 bp) from pCR2.1-gcaD were gel-purified froma 0.8% agarose-0.5X TBE buffer gel using the QIAquick DNA Extraction Kitaccording to the manufacturer's instructions. These two purifiedfragments were ligated together with T4 DNA ligase and the ligation mixwas used to transform E. coli SURE competent cells. Transformants wereselected on 2X YT agar plates supplemented with 100 μg of ampicillin perml at 37° C.

[0239] Plasmids were purified from several transformants using a QIAGENrobot according to the manufacturer's instructions and analyzed by BamHlplus Hpal digestion. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The correct plasmid was identified by the presence of anapproximately 1400 bp BamHl/Hpal gtaB fragment and was designated pHA6(FIG. 12).

[0240] Plasmids pHA6 and pCR2.1-sehasA-(Example 1, FIG. 3) were digestedwith Sacl plus Kpnl. The digestions were resolved on a 0.8% agarose-0.5XTBE buffer gel. The larger vector fragment (approximately 10,200 bp)from pHA6 and the smaller hasA fragment (approximately 1,300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5X TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml. Plasmids were purifiedfrom several transformants using a QIAGEN robot according to themanufacturer's instructions and analyzed by Sacl plus Kpnl digestion.The digestions were resolved on a 0.8% agarose-0.5X TBE buffer gel. Thecorrect plasmid was identified by the presence of an approximately 1300bp Sacl/Kpnl hasA fragment and was designated pHA7 (FIG. 13).

Example 5: Construction of Bacillus subtilis RB161

[0241] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdigested with Sacl. The digested plasmid was then purified using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with Notl. The largest plasmidfragment of approximately 6800 bp was gel-purified using a QIAquick DNAGel Extraction Kit from a 0.8% agarose-0.5X TBE gel according to themanufacturer's instructions (QIAGEN, Valencia, Calif.). The recoveredvector DNA was then ligated with the DNA insert described below.

[0242] Plasmid pHA3 (Example 2, FIG. 9) was digested with Sacl. Thedigested plasmid was then purified as described above, and finallydigested with Notl. The smallest plasmid fragment of approximately 3800bp was gel-purified as described above. The recovered vector and DNAinsert were ligated using the Rapid DNA Cloning Kit (Roche AppliedScience; Indianapolis, Ind.) according to the manufacturer'sinstructions. Prior to transformation in Bacillus subtilis, the ligationdescribed above was linearized using Scal to ensure double cross-overintegration in the chromosome rather than single cross-over integrationin the chromosome. Competent cells of Bacillus subtilis 168Δ4 weretransformed with the ligation products digested with Scal. Bacillussubtilis 168Δ4 is derived from the Bacillus subtilis type strain 168(BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) and hasdeletions in the spollAC, aprE, nprE, and amyE genes. The deletion ofthese four genes was performed essentially as described for Bacillussubtilis Al 64Δ5, which is described in detail in U.S. Pat. No.5,891,701.

[0243] Bacillus subtilis chloramphenicol-resistant transformants wereselected at 34° C. after 16 hours of growth on Tryptose blood agar base(TBAB) plates supplemented with 5 μg of chloramphenicol per ml. Toscreen for integration of the plasmid by double cross-over at the amyElocus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml. Integration of theplasmid by double cross-over at the amyE locus does not incorporate theneomycin resistance gene and therefore renders the strain neomycinsensitive. Isolates were also patched onto minimal plates to visualizewhether or not these were producing hyaluronic acid. Hyaluronic acidproducing isolates have a “wet” phenotype on minimal plates. Using thisplate screen, chloramphenicol resistant and neomycin sensitive “wet”transformants (due to hyaluronic acid production) were isolated at 37°C.

[0244] Genomic DNA was isolated from the “wet”, chloramphenicolresistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformantsusing a QIAGEN tip-20 column (QIAGEN, Valencia, Calif.) according to themanufacturer's instructions. PCR amplifications were performed on thesetransformants using the synthetic oligonucleotides below, which arebased on the hasA, tuaD, and gtaB gene sequences, to confirm thepresence and integrity of these genes in the operon of the Bacillussubtilis transformants.

[0245] The amplification reactions (25 μl) were composed ofapproximately 50 ng of genomic DNA of the Bacillus subtilis 168Δ4transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1X PCR Buffer II, 3 mM MgCl₂, and 0.625 units of AmpliTaqGold™ DNA polymerase. The reactions were incubated in a RoboCycler 40Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for2 minutes; and a final cycle at 72° C. for 7 minutes.

[0246] Primers 3 and 8 were used to confirm the presence of the hasAgene, primers 3 and 16 to confirm the presence of the tuaD gene, andprimers 3 and 22 to confirm the presence of the gtaB gene. The Bacillussubtilis 168Δ4 hasA/tuaD/gtaB integrant was designated Bacillus subtilisRB158.

[0247] Genomic DNA was isolated from Bacillus subtilis RB158 using aQIAGEN tip-20 column according to the manufacturer's instructions, andwas used to transform competent Bacillus subtilis A164Δ5 (deleted at thespollAC, aprE, nprE, amyE, and srfC genes; see U.S. Pat. No. 5,891,701).Transformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. A Bacillus subtilis A164Δ5hasA/tuaD/gtaB integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB161.

Example 6: Construction of Bacillus subtilis RB163

[0248] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdigested with Sacl. The digested plasmid was then purified using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with Notl. The largest plasmidfragment of approximately 6,800 bp was gel-purified using a QIAquick DNAGel Extraction Kit from a 0.8% agarose-0.5X TBE gel according to themanufacturer's instructions. The recovered vector DNA was then ligatedwith the DNA insert described below.

[0249] Plasmid pHA7 (Example 4, FIG. 13) was digested with Sacl. Thedigested plasmid was then purified as described above, and finallydigested with Notl. The smallest plasmid fragment of approximately 4,300bp was gel-purified as described above. The recovered vector and DNAinsert were ligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. Prior to transformation in Bacillussubtilis, the ligation described above was linearized using Scal toensure double cross-over integration in the chromosome rather thansingle cross-over integration in the chromosome. Bacillus subtilis 168Δ4competent cells were transformed with the ligation digested with therestriction enzyme Scal.

[0250] Bacillus subtilis chloramphenicol-resistant transformants wereselected on TBAB plates supplemented with 5 μg of chloramphenicol per mlat 37° C. To screen for integration of the plasmid by double cross-overat the amyE locus, Bacillus subtilis primary transformants were patchedon TBAB plates supplemented with 6 μg of neomycin per ml and on TBABplates supplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive “wet” transformants(due to hyaluronic acid production).

[0251] Genomic DNA was isolated from the “wet”, chloramphenicolresistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformantsusing a QIAGEN tip-20 column according to the manufacturer'sinstructions. PCR amplifications were performed on these transformantsusing primers 3, 8, 16, 22 and primer 30 (Example 1) to confirm thepresence and integrity of these genes in the operon of the Bacillussubtilis transformants. The amplification reactions (25 μl) werecomposed of approximately 50 ng of genomic DNA of the Bacillus subtilis168Δ4 transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1X PCR buffer, 3 mM MgCl₂, and 0.625 units of AmpliTaqGold™ DNA polymerase. The reactions were incubated in a RoboCycler 40Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for2 minutes; and a final cycle at 72° C. for 7 minutes.

[0252] Primers 3 and 8 were used to confirm the presence of the hasAgene, primers 3 and 16 to confirm the presence of the tuaD gene, primers3 and 22 to confirm the presence of the gtaB gene, and primers 3 and 30to confirm the presence of the gcaD gene. The Bacillus subtilis 168Δ4hasA/tuaD/gcaD integrant was designated Bacillus subtilis RB160.

[0253] Genomic DNA was isolated from Bacillus subtilis RB160 using aQIAGEN tip-20 column according to the manufacturer's instructions, andwas used to transform competent Bacillus subtilis A164Δ5. Transformantswere selected on TBAB plates containing 5 μg of chloramphenicol per ml,and grown at 37° C. for 16 hours. The Bacillus subtilis A164Δ5hasA/tuaD/gcaD integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB163.

Example 7: Construction of Bacillus subtilis TH-1

[0254] The hyaluronan synthase (has) operon was obtained fromStreptococcus equisimilis using the following procedure. The has operonis composed of the hasA, hasB, hasC, and hasD genes. Approximately 20 μgof Streptococcus equisimilis D181 (Kumari and Weigel, 1997, Journal ofBiological Chemistry 272: 32539-32546) chromosomal DNA was digested withHindlll and resolved on a 0.8% agarose-0.5X TBE gel. DNA in the 3-6 kbrange was excised from the gel and purified using the QIAquick DNA GelExtraction Kit according to the manufacturer's instructions. Therecovered DNA insert was then ligated with the vector DNA describedbelow.

[0255] Plasmid pUC18 (2 μg) was digested with Hindlll and the 5′protruding ends were dephosphorylated with shrimp alkaline phospataseaccording to the manufacturer's instructions (Roche Applied Science;Indianapolis, Ind.). The dephosphorylated vector and DNA insert wereligated using the Rapid DNA Cloning Kit according to the manufacturer'sinstructions. The ligation was used to transform E. coli XL10 Gold Kancompetent cells (Stratagene, Inc., La Jolla, Calif.). Cells were platedonto Luria broth plates (100 μg/ml ampicillin) and incubated overnightat 37° C. Five plates containing approximately 500 colonies/plate wereprobed with oligo 952-55-1, shown below, which is a 54 bp sequenceidentical to the coding strand near the 3′ end of the Streptococcusequisimilis D181 hasA gene (nucleotides 1098-1151 with respect to the Aresidue of the ATG translation start codon). Primer 31:5′-GTGTCGGAACATTCATTACATGCTTAAGCACCCGCTGTCCTTCTTGTTATCTCC-3′ (SEQ ID NO:39)

[0256] The oligonucleotide probe was DIG-labeled using the DIGOligonucleotide 3′-end Labeling Kit according to the manufacturer'sinstructions (Roche Applied Science; Indianapolis, Ind.). Colonyhybridization and chemiluminescent detection were performed as describedin “THE DIG SYSTEM USER'S GUIDE FOR FILTER HYBRIDIZATION”, BoehringerMannheim GmbH.

[0257] Seven colonies were identified that hybridized to the probe.Plasmid DNA from one of these transformants was purified using a QIAGENrobot (QIAGEN, Valencia, Calif.) according to the manufacturer'sinstructions, digested with Hindlll, and resolved on a 0.8% agarose gelusing 0.5X TBE buffer. The DNA insert was shown to be approximately 5 kbin size. This plasmid was designated pMRT106 (FIG. 14).

[0258] The DNA sequence of the cloned fragment was determined using theEZ::TN™ <TET-1> Insertion Kit according to the manufacturer'sinstructions (Epicenter Technologies, Madison, Wis.). The sequencingrevealed that the cloned DNA insert contained the last 1156 bp of theStreptococcus equisimilis hasA gene followed by three other genesdesignated hasB, hasC, and hasD; presumably all four genes are containedwithin a single operon and are therefore co-transcribed. TheStreptococcus equisimilis hasB gene is contained in nucleotides1411-2613 (SEQ ID NOs: 40 [DNA sequence] and 41 [deduced amino acidsequence]) of the fragment, and Streptococcus equisimilis hasC gene innucleotides 2666-3565 (SEQ ID NOs: 42 [DNA sequence] and 43 [deducedamino acid sequence]) of the fragment, and Streptococcus equisimilishasD gene in nucleotides 3735-5114 (SEQ ID NOs: 44 [DNA sequence] and 45[deduced amino acid sequence]) of the fragment.

[0259] The polypeptides encoded by the Streptococcus equisimilis hasBand hasC genes show some homology to those encoded by the hasB and hasCgenes, respectively, from the Streptococcus pyogenes has operon sequence(Ferretti et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98 (8),4658-4663). The degree of identity was determined by the Clustal method(Higgins, 1989, CABIOS 5: 151-153) using using the Vector NTI AlignXsoftware (Informax Inc., Bethesda, Md.) with the following defaults:pairwise alignment, gap opening penalty of 10, gap extension penalty of0.1, and score matrix: blosum62mt2.

[0260] Amino acid sequence comparisons showed that the Streptococcusequisimilis HasB protein has 70% identity to the HasB protein fromStreptococcus uberis (SEQ ID NO: 105); the Streptococcus equisimilisHasC protein has 91% identity to the HasC protein from Streptococcuspyogenes (SEQ ID NO: 99); and the Streptococcus equisimilis HasD proteinhas 73% identity to the GlmU protein (a putative UDP-N-acetylglucosaminepyrophosphorylase) of Streptococcus pyogenes (accession # Q8P286). TheStreptococcus equisimilis hasD gene encodes a polypeptide that shows50.7% identity to the UDP-N-acetyl-glucosamine pyrophosphorylase enzymeencoded by the gcaD gene of Bacillus subtilis.

[0261] Plasmid pHA5 (Example 3, FIG. 11) was digested with Hpal andBamHl. The digestion was resolved on a 0.8% agarose gel using 0.5X TBEbuffer and the larger vector fragment (approximately 11,000 bp) wasgel-purified using the QIAquick DNA Extraction Kit according to themanufacturer's instructions. Plasmid pMRT106 was digested with Hindlll,the sticky ends were filled in with Klenow fragment, and the DNA wasdigested with BamHl. The digestion was resolved on a 0.8% agarose gelusing 0.5X TBE buffer and the smaller insert fragment (approximately1000 bp, the last ⅔ of the Streptococcus equisimilis hasD gene) wasgel-purified using the QIAquick DNA Extraction kit according to themanufacturer's instructions.

[0262] The two purified fragments were ligated together with T4 DNAligase and the ligation mix was transformed into E. coli SURE competentcells. Transformants were selected on 2X YT agar plates supplementedwith 100 μg of ampicillin per ml at 37° C.

[0263] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby BamHl plus Notl digestion on a 0.8% agarose gel using 0.5X TBEbuffer. The correct plasmid was identified by the presence of anapproximately 1,100 bp BamHl/Notl hasD fragment and was designated pHA8(FIG. 15). This plasmid was digested with Hindlll and ligated togetherwith T4 DNA ligase and the ligation mix was transformed into E. coliSURE competent cells. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml. Plasmid DNA was purifiedfrom several transformants using a QIAGEN robot according to themanufacturer's instructions and analyzed by Hindlll digestion on a 0.8%agarose gel using 0.5X TBE buffer. The correct plasmid was identified bythe presence of a single band of approximately 9,700 bp and wasdesignated pHA9 (FIG. 16).

[0264] Plasmid pHA9 was digested with Sacl and Notl. The digestion wasresolved on a 0.8% agarose gel using 0.5X TBE buffer and the smallerfragment of approximately 2,500 bp was gel-purified using the QIAquickDNA Extraction kit according to the manufacturer's instructions. PlasmidpDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with Sacland Notl. The digestion was resolved on a 0.8% agarose gel using 0.5XTBE buffer and the larger vector fragment of approximately 6,800 bp wasgel-purified using the QIAquick DNA Extraction kit according to themanufacturer's instructions. The two purified fragments were ligatedtogether with T4 DNA ligase and the ligation mix was transformed into E.coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.).Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml.

[0265] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby Sall plus Hindlll digestion on a 0.8% agarose gel using 0.5X TBEbuffer. The correct plasmid was identified by the presence of anapproximately 1600 bp Sall/Hindlll fragment and was designated pHA10(FIG. 17).

[0266] Plasmid pHA10 was digested with Hindlll and BamHl. The digestionwas resolved on a 0.8% agarose gel using 0.5X TBE buffer and the largervector fragment (approximately 8100 bp) was gel-purified using theQIAquick DNA Extraction kit according to the manufacturer'sinstructions. Plasmid pMRT106 was digested with Hindlll and BamHl. Thedigestion was resolved on a 0.8% agarose gel using 0.5X TBE buffer andthe larger insert fragment of approximately 4,100 bp was gel-purifiedusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform Bacillus subtilis168Δ4. Transformants were selected on TBAB agar plates supplemented with5 μg of chloramphenicol per ml at 37° C. Approximately 100 transformantswere patched onto TBAB supplemented with chloramphenicol (5 μg/ml) andTBAB supplemented with neomycin (10 μg/ml) to score chloramphenicolresistant, neomycin sensitive colonies; this phenotype is indicative ofa double crossover into the amyE locus. A few such colonies wereidentified, all of which exhibited a “wet” phenotype indicating thathyaluronic acid was being produced. One colony was chosen and designatedBacillus subtilis 168Δ4::scBAN/se hasA/hasB/hasC/hasD.

[0267] Genomic DNA was isolated from Bacillus subtilis 168Δ4::scBAN/sehasA/hasB/hasC/hasD using a QIAGEN tip-20 column according to themanufacturer's instructions, and used to transform competent Bacillussubtilis A164Δ5. Transformants were selected on TBAB plates containing 5μg of chloramphenicol per ml, and grown at 37° C. for 16 hours. TheBacillus subtilis A164Δ5 hasA/hasB/hasC/hasD integrant was identified byits “wet” phenotype and designated Bacillus subtilis TH-1.

Example 8: Construction of Bacillus subtilis RB184

[0268] The hasA gene from Streptococcus equisimilis (Example 1) and tuaDgene (a Bacillus subtilis hasB homologue) (Example 1) were cloned to beunder the control of a short “consensus” amyQ (scBAN) promoter (U.S.Pat. No. 5,955,310).

[0269] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdigested with Sacl. The digested plasmid was then purified using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with Notl. The largest plasmidfragment of approximately 6,800 bp was gel-purified from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Gel Extraction Kit accordingto the manufacturer's instructions. The recovered vector DNA was thenligated with the DNA insert described below.

[0270] Plasmid pHA5 (Example 3, FIG. 11) was digested with Hpal. Thedigested plasmid was then purified as described above, and finallydigested with Xbal. The double-digested plasmid was then blunted byfirst inactivating Xbal at 85° C. for 30 minutes. Blunting was performedby adding 0.5 μl of 10 mM each dNTPs, 1 μl of 1 U/μl T4 DNA polymerase(Roche Applied Science; Indianapolis, Ind.) and incubating at 11° C. for10 minutes. Finally the polymerase was inactivated by incubating thereaction at 75° C. for 10 minutes. The largest plasmid fragment ofapproximately 11,000 bp was then gel-purified as described above andreligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. The ligation mix was transformed into E.coli SURE competent cells. Transformants were selected on 2X YT agarplates supplemented with 100 μg of ampicillin per ml at 37° C. PlasmidDNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by Scaldigestion on a 0.8% agarose gel using 0.5X TBE buffer. The correctplasmid was identified by the presence of an approximately 11 kbfragment and was designated pRB157 (FIG. 18).

[0271] pRB157 was digested with Sacl. The digested plasmid was thenpurified using a QIAquick DNA Purification Kit according to themanufacturer's instructions, and finally digested with Notl. Thesmallest plasmid fragment of approximately 2,632 bp was gel-purifiedusing a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions. The recovered DNA insertwas then ligated with the vector DNA described above.

[0272] Prior to transformation in Bacillus subtilis, the ligationdescribed above was linearized using Scal to ensure double cross-overintegration in the chromosome rather than single cross-over integrationin the chromosome. Bacillus subtilis 168Δ4 competent cells weretransformed with the ligation digested with the restriction enzyme Scal.

[0273] Bacillus subtilis chloramphenicol-resistant transformants wereselected on TBAB plates supplemented with 5 μg of chloramphenicol perml. To screen for integration of the plasmid by double cross-over at theamyE locus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive “wet” transformants(due to hyaluronic acid production).

[0274] Genomic DNA was isolated from the “wet”, chloramphenicolresistant, and neomycin sensitive Bacillus subtilis 168Δ4 transformantsusing a QIAGEN tip-20 column according to the manufacturer'sinstructions. PCR amplifications were performed on these transformantsusing primers 3, 8, and 16 (Example 1) to confirm the presence andintegrity of hasA and tuaD in the operon of the Bacillus subtilistransformants. The amplification reactions (25 μl) were composed ofapproximately 50 ng of genomic DNA of the Bacillus subtilis 168Δ4transformants, 0.5 μM of each primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1X PCR buffer, 3 mM MgCl₂, and 0.625 units of AmpliTaq Gold™DNA polymerase. The reactions were incubated in a RoboCycler 40Temperature Cycler programmed for one cycle at 95° C. for 9 minutes; 30cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for2 minutes; and a final cycle at 72° C. for 7 minutes.

[0275] Primers 3 and 8 were used to confirm the presence of the hasAgene and primers 3 and 16 to confirm the presence of the tuaD gene. ABacillus subtilis 168Δ4 hasA/tuaD integrant was designated Bacillussubtilis RB183.

[0276] Bacillus subtilis RB183 genomic DNA was also used to transformcompetent Bacillus subtilis A164Δ5. Transformants were selected on TBABplates containing 5 μg of chloramphenicol per ml, and grown at 37° C.for 16 hours. The Bacillus subtilis A164Δ5 hasA/tuaD integrant wasidentified by its “wet” phenotype and designated Bacillus subtilisRB184.

Example 9: Construction of Bacillus subtilis RB187

[0277] Bacillus subtilis RB161 was made competent and transformed withthe cat deletion plasmid pRB115 (Widner et al., 2000, Journal ofIndustrial Microbiology & Biotechnology 25: 204-212). Selection fordirect integration into the chromosome was performed at thenon-permissive temperature of 45° C. using erythromycin (5 μg/ml)selection. At this temperature, the pE194 origin of replication isinactive. Cells are able to maintain erythromycin resistance only byintegration of the plasmid into the cat gene on the bacterialchromosome. These so-called “integrants” were maintained at 45° C. toensure growth at this temperature with selection. To allow for loss or“looping out” of the plasmid, which will result in the deletion of mostof the cat gene from the chromosome, the integrants were grown inLuria-Bertani (LB) medium without selection at the permissivetemperature of 34° C. for many generations. At this temperature thepE194 origin of replication is active and promotes excision of theplasmid from the genome (Molecular Biological Methods for Bacillus,edited by C. R. Harwood and S. M. Cutting, 1990, John Wiley and SonsLtd.).

[0278] The cells were then plated on non-selective LB agar plates andcolonies which contained deletions in the cat gene and loss of thepE194-based replicon were identified by the following criteria: (1)chloramphenicol sensitivity indicated the presence of the cat deletion;(2) erythromycin sensitivity indicated the absence of the erythromycinresistance gene encoded by the vector pRB115; and (3) PCR confirmed thepresence of the cat deletion in the strain of interest. PCR wasperformed to confirm deletion of the cat gene at the amyE locus by usingprimers 32 and 33: Primer 32: (SEQ ID NO: 46)5′-GCGGCCGCGGTACCTGTGTTACACCTGTT-3′ Primer 33: (SEQ ID NO: 47)5′-GTCAAGCTTAATTCTCATGTTTGACAGCTTATCATCGG-3′

[0279] Chromosomal DNA from potential deletants was isolated using theREDextract-N-Amp™ Plant PCR kits (Sigma Chemical Company, St. Louis,Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl ofExtraction Solution (Sigma Chemical Company, St. Louis, Mo.), incubatedat 95° C. for 10 minutes, and then diluted with an equal volume ofDilution Solution (Sigma Chemical Company, St. Louis, Mo.). PCR wasperformed using 4 μl of extracted DNA in conjunction with theREDextract-N-Amp PCR Reaction Mix and the desired primers according tothe manufacturer's instructions, with PCR cycling conditions describedin Example 5. PCR reaction products were visualized in a 0.8%agarose-0.5X TBE gel. The verified strain was named Bacillus subtilisRB187.

Example 10: Construction of Bacillus subtilis RB192

[0280] Bacillus subtilis RB184 was made unmarked by deleting thechloramphenicol resistance gene (cat gene). This was accomplished usingthe method described previously in Example 9. The resultant strain wasdesignated Bacillus subtilis RB192.

Example 11: Construction of Bacillus subtilis RB194

[0281] Bacillus subtilis RB194 was constructed by deleting the cypXregion of the chromosome of Bacillus subtilis RB187 (Example 9). ThecypX region includes the cypX gene which encodes a cytochrome P450-likeenzyme that is involved in the synthesis of a red pigment duringfermentation. In order to delete this region of the chromosome plasmidpMRT086 was constructed.

[0282] The region of the chromosome which harbors the cypX-yvmC andyvmB-yvmA operons was PCR amplified from Bacillus subtilis BRG-1 as asingle fragment using primers 34 and 35. Bacillus subtilis BRG1 isessentially a chemically mutagenized isolate of an amylase-producingstrain of Bacillus subtilis which is based on the Bacillus subtilisA164Δ5 genetic background that was described in Example 5. The sequenceof this region is identical to the published sequence for the Bacillussubtilis 168 type strain. Primer 34: 5′-CATGGGAGAGACCTTTGG-3′ (SEQ IDNO: 48) Primer 35: 5′-GTCGGTCTTCCATTTGC-3′ (SEQ ID NO: 49)

[0283] The amplification reactions (50 μl) were composed of 200 ng ofBacillus subtilis BRG-1 chromosomal DNA, 0.4 μM each of primers 34 and35, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1X Expand™ High Fidelitybuffer (Roche Applied Science; Indianapolis, Ind.) with 1.5 mM MgCl₂,and 2.6 units of Expand™ High Fidelity PCR System enzyme mix (RocheApplied Science; Indianapolis, Ind.). Bacillus subtilis BRG-1chromosomal DNA was obtained using a QIAGEN tip-20 column according tothe manufacturer's instructions. Amplification reactions were performedin a RoboCycler 40 thermacycler (Stratagene, Inc, La Jolla, Calif.)programmed for 1 cycle at 95° C. for 3 minutes; 10 cycles each at 95° C.for 1 minute, 58° C. for 1 minute, and 68° C. for 4 minutes; 20 cycleseach at 95° C. for 1 minute, 58° C. for 1 minute, 68° C. for 4 minutesplus 20 seconds per cycle, followed by 1 cycle at 72° C. for 7 minutes.Reaction products were analyzed by agarose gel electrophoresis using a0.8% agarose gel using 0.5X TBE buffer.

[0284] The resulting fragment comprising the cypX-yvmC and yvmB-yvmAoperons was cloned into pCR2.1 using the TA-TOPO Cloning Kit andtransformed into E. coli OneShot™ cells according to the manufacturer'sinstructions (Invitrogen, Inc., Carlsbad, Calif.). Transformants wereselected on 2X YT agar plates supplemented with 100 μg of ampicillin perml. Plasmid DNA from several transformants was isolated using QIAGENtip-20 columns according to the manufacturer's instructions and verifiedby DNA sequencing with M13 (−20) forward, M13 reverse and primers 36 to51 shown below. The resulting plasmid was designated pMRT084 (FIG. 19).Primer 36: 5′-CGACCACTGTATCTTGG-3′ (SEQ ID NO: 50) Primer 37:5′-GAGATGCCAAACAGTGC-3′ (SEQ ID NO: 51) Primer 38:5′-CATGTCCATCGTGACG-3′ (SEQ ID NO: 52) Primer 39:5′-CAGGAGCATTTGATACG-3′ (SEQ ID NO: 53) Primer 40:5′-CCTTCAGATGTGATCC-3′ (SEQ ID NO: 54) Primer 41:5′-GTGTTGACGTCAACTGC-3′ (SEQ ID NO: 55) Primer 42:5′-GTTCAGCCTTTCCTCTCG-3′ (SEQ ID NO: 56) Primer 43:5′-GCTACCTTCTTTCTTAGG-3′ (SEQ ID NO: 57) Primer 44:5′-CGTCAATATGATCTGTGC-3′ (SEQ ID NO: 58) Primer 45:5′-GGAAAGAAGGTCTGTGC-3′ (SEQ ID NO: 59) Primer 46:5′-CAGCTATCAGCTGACAG-3′ (SEQ ID NO: 60) Primer 47:5′-GCTCAGCTATGACATATTCC-3′ (SEQ ID NO: 61) Primer 48:5′-GATCGTCTTGATTACCG-3′ (SEQ ID NO: 62) Primer 49:5′-AGCTTTATCGGTGACG-3′ (SEQ ID NO: 63) Primer 50: 5′-TGAGCACGATTGCAGG-3′(SEQ ID NO: 64) Primer 51: 5′-CATTGCGGAGACATTGC-3′ (SEQ ID NO: 65)

[0285] Plasmid pMRT084 was digested with Bsgl to delete most of thecypX-yvmC and yvmB-yvmA operons, leaving about 500 bases at each end.The digested Bsgl DNA was treated with T4 DNA polymerase. Plasmid pECC1(Youngman et al., 1984, Plasmid 12: 1-9) was digested with Smal. Afragment of approximately 5,100 bp from pMRT084 and a fragment ofapproximately 1,600 bp fragment from pECC1 were isolated from a 0.8%agarose-0.5X TBE gel using the QIAquick DNA Extraction Kit according tothe manufacturer's instructions, ligated together, and transformed intoE. coli XL1 Blue cells according to the manufacturer's instructions(Stratagene, Inc., La Jolla, Calif.). Transformants were selected on 2XYT agar plates supplemented with 100 μg of ampicillin per ml.Transformants carrying the correct plasmid with most of the cypX-yvmCand yvmB-yvmA operons deleted were identified by PCR amplification usingprimers 52 and 53. PCR amplification was conducted in 50 μl reactionscomposed of 1 ng of plasmid DNA, 0.4 μM of each primer, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5units of AmpliTaq Gold™ DNA polymerase. The reactions were performed ina RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCRproduct was visualized using a 0.8% agarose-0.5X TBE gel. This constructwas designated pMRT086 (FIG. 20). Primer 52:5′-TAGACAATTGGAAGAGAAAAGAGATA-3′ (SEQ ID NO: 66) Primer 53:5′-CCGTCGCTATTGTAACCAGT-3′ (SEQ ID NO: 67)

[0286] Plasmid pMRT086 was linearized with Scal and transformed intoBacillus subtilis RB128 competent cells in the presence of 0.2 μg ofchloramphenicol per ml. Transformants were selected on TBAB platescontaining 5 μg of chloramphenicol per ml after incubation at 37° C. for16 hours. Chromosomal DNA was prepared from several transformants usinga QIAGEN tip-20 column according to the manufacturer's instructions.Chloramphenicol resistant colonies were screened by PCR for deletion ofthe cypX-yvmC and yvmB-yvmA operons via PCR using primers 36 and 52, 36and 53, 37 and 52, and 37 and 53. PCR amplification was conducted in 50μl reactions composed of 50 ng of chromosomal DNA, 0.4 μM of eachprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1X PCR Buffer II with2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNA polymerase. Thereactions were performed in a RoboCycler 40 thermacycler programmed for1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for7 minutes. The PCR products were visualized using a 0.8% agarose-0.5XTBE gel. The resulting Bacillus subtilis RB128 cypX-yvmC and yvmB-yvmAdeleted strain was designated Bacillus subtilis MaTa17.

[0287] Competent cells of Bacillus subtilis RB187 (Example 9) weretransformed with genomic DNA from Bacillus subtilis MaTa17. Genomic DNAwas obtained from this strain using a QIAGEN tip-20 column according tothe manufacturer's instructions. Bacillus subtilis chloramphenicolresistant transformants were selected on TBAB plates supplemented with 5μg of chloramphenicol per ml at 37° C. Primary transformants werestreaked for single colony isolations on TBAB plates containing 5 μg ofchloramphenicol per ml at 37° C. The resulting cypX-yvmC and yvmB-yvmAdeleted strain was designated Bacillus subtilis RB194.

Example 12: Construction of Bacillus subtilis RB197

[0288] Bacillus subtilis RB197 is very similar to Bacillus subtilisRB194, the only difference being that RB197 contains a smaller deletionin the cypX region: only a portion of the cypX gene is deleted in thisstrain to generate a cypX minus phenotype. In order to accomplish thistask a plasmid, pMRT122, was constructed as described below.

[0289] Plasmid pCJ791 (FIG. 21) was constructed by digestion of plasmidpSJ2739 (WO 96/23073) with EcoRI/Hindlll and ligation to a fragmentcontaining a deleted form of the wprA gene (cell wall serine protease)from Bacillus subtilis. The 5′ region of wprA was amplified usingprimers 54 and 55 see below, and the 3′ region was amplified usingprimers 56 and 57 shown below from chromosomal DNA obtained fromBacillus subtilis DN1885 (Diderichsen et al., 1990, Journal ofBacteriology 172: 4315-4321). PCR amplification was conducted in 50 μlreactions composed of 1 ng of Bacillus subtilis DN1885 chromosomal DNA,0.4 μMeach of primers 39 and 40, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycleseach at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 7 minutes.

[0290] The 5′ and 3′ wprA PCR fragments were linked by digestion withBg/ll followed by ligation, and PCR amplification was performed on theligation mixture fragments using primers 54 and 57. PCR amplificationwas conducted in 50 μl reactions composed of 1 ng of the ligatedfragment, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycleseach at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized using a 0.8% agarose-0.5X TBE gel. The resulting PCR fragmentwas cloned into pSJ2739 as an EcoRI/Hindlll fragment, resulting inplasmid pCJ791 (FIG. 21). Transformants were selected on TBAB-agarplates supplemented with 1 μg of erythromycin and 25 μg of kanamycin perml after incubation at 28° C. for 24-48 hours. Plasmid DNA from severaltransformants was isolated using QIAGEN tip-20 columns according to themanufacturer's instructions and verified by PCR amplification withprimers 54 and 57 using the conditions above. Primer 54: (SEQ ID NO: 68)5′-GGAATTCCAAAGCTGCAGCGGCCGGCGCG-3′ Primer 55: (SEQ ID NO: 69)5′-GAAGATCTCGTATACTTGGCTTCTGCAGCTGC-3′ Primer 56: (SEQ ID NO: 70)5′-GAAGATCTGGTCAACAAGCTGGAAAGCACTC-3′ Primer 57: (SEQ ID NO: 71)5′-CCCAAGCTTCGTGACGTACAGCACCGTTCCGGC-3′

[0291] The amyL upstream sequence and 5′ coding region from plasmidpDN1981 (U.S. Pat. No. 5,698,415) were fused together by SOE using theprimer pairs 58/59 and 60/61 shown below. The resulting fragment wascloned into vector pCR2.1 to generate plasmid pMRT032 as follows. PCRamplifications were conducted in triplicate in 50 μl reactions composedof 1 ng of pDN1981 DNA, 0.4 μM each of appropriate primers, 200 μM eachof dATP, dCTP, dGTP, and dTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and2.5 units of AmpliTaq Gold™ DNA polymerase. The reactions were performedin a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 9minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes.The PCR product was visualized in a 0.8% agarose-0.5X TBE gel. Theexpected fragments were approximately 530 and 466 bp, respectively. Thefinal SOE fragment was generated using primer pair 59/60 and cloned intopCR2.1 vector using the TA-TOPO Cloning Kit. Transformants were selectedon 2X YT agar plates supplemented with 100 μg/ml ampicillin afterincubation at 37° C. for 16 hours. Plasmid DNA from severaltransformants was isolated using QIAGEN tip-20 columns according to themanufacturer's instructions and verified by DNA sequencing with M13(−20) forward and M13 reverse primers. The plasmid harboring the amyLupstream sequence/5′ coding sequence fusion fragment was designatedpMRT032 (FIG. 22). Primer 58:5′-CCTTAAGGGCCGAATATTTATACGGAGCTCCCTGAAACAACAAAAACGGC-3′ (SEQ ID NO: 72)Primer 59: 5′-GGTGTTCTCTAGAGCGGCCGCGGTTGCGGTCAGC-3′ (SEQ ID NO: 73)Primer 60: 5′-GTCCTTCTTGGTACCTGGAAGCAGAGC-3′ (SEQ ID NO: 74) Primer 61:5′-GTATAAATATTCGGCCCTTAAGGCCAGTACCATTTTCCC-3′ (SEQ ID NO: 75)

[0292] Plasmid pNNB194 (pSK⁺/pE194; U.S. Pat. No. 5,958,728) wasdigested with Nsil and Notl, and plasmid pBEST501 (Itaya et al. 1989Nucleic Acids Research 17: 4410) was digested with Pstl and Notl. The5,193 bp vector fragment from pNNB194 and the 1,306 bp fragment bearingthe neo gene from pBEST501 were isolated from a 0.8% agarose-0.5X TBEgel using a QIAquick DNA Purification Kit according to themanufacturer's instructions. The isolated fragments were ligatedtogether and used to transform E. coli SURE competent cells according tothe manufacturer's instructions. Ampicillin-resistant transformants wereselected on 2X YT plates supplemented with 100 μg of ampicillin per ml.Plasmid DNA was isolated from one such transformant using the QIAGENPlasmid Kit (QIAGEN Inc., Valencia, Calif.), and the plasmid wasverified by digestion with Nsil and Notl. This plasmid was designatedpNNB194neo (FIG. 23).

[0293] Plasmid pNNB194neo was digested with Sacl/Notl and treated withT4 DNA polymerase and dNTPs to generate blunt ends using standardprotocols. Plasmid pPL2419 (U.S. Pat. No. 5,958,728) was digested withEcl13611. The 6,478 bp vector fragment from pNNB194neo and the 562 bpfragment bearing oriT from pPL2419 were isolated from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Purification Kit according tothe manufacturer's instructions. The gel-purified fragments were ligatedtogether and used to transform E. coli SURE cells according to themanufacturer's instructions. Ampicillin-resistant transformants wereselected on 2X YT plates supplemented with 100 μg of ampicillin per mlat 37° C. Plasmid DNA was isolated from one such transformant using theQIAGEN Plasmid Kit, and the plasmid was verified by digestion with NSil,Sacl, and Bscl. This plasmid was designated pNNB194neo-oriT (FIG. 24).

[0294] Plasmid pNNB194neo-oriT was digested with BamHl and treated withT4 DNA polymerase and dNTPs to generate blunt ends using standardprotocols. The digested plasmid was gel-purified from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Purification Kit according tothe manufacturer's instructions. The purified plasmid was treated withT4 DNA ligase and used to transform E. coli SURE cells according to themanufacturer's instructions. Ampicillin-resistant transformants wereselected on 2X YT plates supplemented with 100 μg of ampicillin per mlat 37° C. Plasmid DNA was isolated from one such transformant using theQIAGEN Plasmid Kit, and disruption of the BamHl site was confirmed bydigestion with BamHl and Scal. The resulting plasmid was designatedpShV3 (FIG. 25).

[0295] Plasmid pShV2.1-amyEΔ (U.S. Pat. No. 5,958,728) was digested withSfil and Notl, and the 8696 bp vector fragment was gel-purified from a0.8% agarose-0.5X TBE gel using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions. In order to insert a BamHlsite between the Sfil and Notl sites of pShV2.1-amyEΔ, a syntheticlinker was constructed as follows: primers 62 and 63 were annealed bymixing 50 μM of each, boiling the mixture, and allowing the mixture tocool slowly. Primer 62: 5′-GGGCCGGATCCGC-3′ (SEQ ID NO: 76) Primer 63:3′-ATTCCCGGCCTAGGCGCCGG-5′ (SEQ ID NO: 77)

[0296] The purified pShV2.1-amyEΔ vector and annealed oligonucleotideswere ligated together and used to transform E. coli SURE competent cellsaccording to the manufacturer's instructions. Chloramphenicol-resistanttransformants were selected on LB plates supplemented with 30 μg ofchloramphenicol per ml at 37° C. Plasmid DNA was isolated from one suchtransformant using the QIAGEN Plasmid Kit, and insertion of the BamHlsite was confirmed by digestion with BamHl. This plasmid was designatedpShV2.1-amyEΔB (FIG. 26).

[0297] Plasmids pShV3 and pShV2.1-amyEΔB were digested withSall/Hindlll. A 7033 bp vector fragment from pShV3 and a 1031 bpfragment bearing amyEΔ from pShV2.1-amyEΔ were gel-purified from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Purification Kit according tothe manufacturer's instructions. The gel-purified fragments were ligatedtogether and used to transform E. coli SURE cells according to themanufacturer's instructions. Ampicillin-resistant transformants wereselected on 2X YT plates supplemented with 100 μg of ampicillin per ml.Plasmid DNA was isolated from one such transformant using the QIAGENPlasmid Kit, and the plasmid was verified by digestion with Sall andHindlll. This plasmid was designated pShV3A (FIG. 27).

[0298] Plasmid pMRT032 was digested with Kpnl/Xbal, filled with Klenowfragment DNA polymerase in the presence of dNTPs, and a fragment ofapproximately 1000 bp was isolated from a 0.8% agarose-0.5X TBE gelusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions. This fragment was cloned into plasmid pShV3a digested withEcoRV, and transformed into E. coli XL1 Blue cells according to themanufacturer's instructions. Transformants were selected on 2X YT agarplates supplemented with 100 μg of ampicillin per ml after incubation at37° C. for 16 hours. Plasmid DNA from several transformants was isolatedusing QIAGEN tip-20 columns according to the manufacturer's instructionsand verified on a 0.8% agarose-0.5X TBE gel by restriction analysis withSacl/Sphl. The resulting plasmid was designated pMRT036 (FIG. 28).

[0299] Plasmid pMRT036 was digested with Sall/Hindlll, filled withKlenow fragment DNA polymerase in the presence of dNTPs, ligated andtransformed into E. coli XL1 Blue cells according to the manufacturer'sinstructions. Transformants were selected on 2X YT-agar platessupplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16hours. Plasmid DNA from several transformants was isolated using QIAGENtip-20 columns according to the manufacturer's instructions and verifiedon a 0.8% agarose-0.5X TBE gel by restriction analysis with Sacl/Xbal,Pstl and Ndel. The resulting plasmid was designated pMRT037 (FIG. 29).

[0300] The scBAN/crylllA stabilizer fragment from plasmidpDG268Δneo-crylllAstab/Sav (U.S. Pat. No. 5,955,310) was isolated from a2% agarose-0.5X TBE gel as a Sfil/Sacl fragment using a QIAquick DNAPurification Kit according to the manufacturer's instructions, ligatedto plasmid pMRT037 digested with Sfil/Sacl, and transformed into E. coliXL1 Blue cells. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml after incubation at 37° C.for 16 hours. Plasmid DNA from several transformants was isolated usingQIAGEN tip-20 columns according to the manufacturer's instructions andverified on a 0.8% agarose-0.5X TBE gel by restriction analysis withPstl. The resulting plasmid was designated pMRT041 (FIG. 30).

[0301] Plasmids pMRT041 and pCJ791 were digested with EcoRI/Hindlll. Afragment of approximately 1300 bp from pMRT041 and a fragment ofapproximately 4500 bp from pCJ791 were isolated from a 0.8% agarose-0.5XTBE gel using a QIAquick DNA Purification Kit according to themanufacturer's instructions, ligated, and transformed into Bacillussubtilis 168Δ4 competent cells. Transformants were selected on TBAB-agarplates supplemented with 1 μg of erythromycin and 25 μg of lincomycinper ml after incubation at 30° C. for 24-48 hours. Plasmid DNA fromseveral transformants was isolated using QIAGEN tip-20 columns accordingto the manufacturer's instructions and verified on a 0.8% agarose-0.5XTBE gel by restriction analysis with Sacl and EcoRI/Hindlll. Theresulting plasmid was designated pMRT064.1 (FIG. 31).

[0302] The Sacl site at position 2666 in plasmid pMRT064.1 was deletedby SOE using primer pairs 64 and 65, and primer pairs 66 and 67 shownbelow. PCR amplification was conducted in 50 μl reactions composed of 1ng of pMRT064.1 DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units ofAmpliTaq Gold™ DNA polymerase. The reactions were performed in aRoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10minutes; 25 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCRproduct was visualized in a 0.8% agarose-0.5X TBE gel. The expectedfragments were approximately 400 and 800 bp, respectively. The finalfragment for cloning back into pMRT064.1 was amplified using primers 64and 67. This fragment was cloned into pCR2.1 vector using the TA-TOPOCloning Kit. Transformants were selected on 2X YT agar platessupplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16hours. Transformants carrying the correct plasmid were verified by DNAsequencing using M13 forward and reverse primers, and primers 65, 67,and 68. This plasmid was designated pMRT068 (FIG. 32), and was furthertransformed into E. coli DM1 cells (Stratagene, Inc., La Jolla, Calif.)according to the manufacturer's instructions. Transformants wereselected on 2X YT agar plates supplemented with 100 μg of ampicillin perml. Primer 64: 5′-GGAAATTATCGTGATCAAC-3′ (SEQ ID NO: 78) Primer 65:5′-GCACGAGCACTGATAAATATG-3′ (SEQ ID NO: 79) Primer 66:5′-CATATTTATCAGTGCTCGTGC-3′ (SEQ ID NO: 80) Primer 67:5′-TCGTAGACCTCATATGC-3′ (SEQ ID NO: 81) Primer 68:5′-GTCGTTAAACCGTGTGC-3′ (SEQ ID NO: 82)

[0303] The Sacl sites at positions 5463 and 6025 in plasmid pMRT064.1were deleted using PCR amplification with primers 69 and 70, and usingthe PCR conditions described above. The resulting fragment was clonedinto pCR2.1 vector using the TA-TOPO Cloning Kit (Invitrogen, Inc.,Carlsbad, Calif.). Transformants were selected on 2X YT-agar platessupplemented with 100 μg of ampicillin per ml after incubation at 37° C.for 16 hours. Transformants carrying the correct plasmid were verifiedby DNA sequencing using M13 forward and reverse primers. This constructwas designated pMRT069 (FIG. 33). Primer 69: (SEQ ID NO: 83)5′-CTAGAGGATCCCCGGGTACCGTGCTCTGCCTTTTAGTCC-3′ Primer 70: (SEQ ID NO: 84)5′-GTACATCGAATTCGTGCTCATTATTAATCTGTTCAGC-3′

[0304] Plasmids pMRT068 and pMRT064.1 were digested with Bcll/Accl. Afragment of approximately 1300 bp from pMRT068 and a fragment ofapproximately 3800 bp from pMRT064.1 were isolated from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Purification Kit according tothe manufacturer's instructions, ligated, and transformed into Bacillussubtilis 168Δ4 competent cells. Transformants were selected on TBAB-agarplates supplemented with 1 μg of erythromycin and 25 μg of lincomycinper ml after incubation at 30° C. for 24-48 hours. Transformantscarrying the correct plasmid were identified on a 0.8% agarose-0.5X TBEgel by restriction analysis with Sacl and EcoRI/Aval. The resultingconstruct was designated pMRT071 (FIG. 34).

[0305] Plasmids pMRT071 and pMRT069 were digested with Aval/EcoRI. The578 bp fragment from pMRT069 and the 4510 bp fragment from pMRT071 wereisolated from a 0.8% agarose-0.5X TBE gel using a QIAquick DNAPurification Kit according to the manufacturer's instructions, ligated,and transformed into Bacillus subtilis 168Δ4 competent cells.Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml after incubation at 30°C. for 24-48 hours. Transformants carrying the correct plasmid wereidentified on a 0.8% agarose-0.5X TBE gel by restriction analysis withSacl. The resulting construct was designated pMRT074 (FIG. 35).

[0306] Plasmid pMRT084 described in Example 11 was digested withSacll/Ndel, treated with T4 DNA polymerase, ligated, and transformedinto E. coli XL1 Blue cells according to the manufacturer'sinstructions. Transformants were selected on 2X YT agar platessupplemented with 100 μg of ampicillin per ml after incubation at 37° C.for 16 hours. Transformants carrying the correct plasmid were identifiedon a 0.8% agarose-0.5X TBE gel by restriction analysis with Dral. Theresulting plasmid was named pMRT120 (FIG. 36).

[0307] Plasmid pMRT074 was digested with Hindlll, treated with Klenowfragment DNA polymerase, and digested with EcoRI. Plasmid pMRT120 wasdigested with EcoRI/Ecl13611. A fragment of approximately 600 bp frompMRT120 and a fragment of approximately 4300 bp from pMRT074 wereisolated from a 0.8% agarose-0.5X TBE gel using a QIAquick DNAPurification Kit according to the manufacturer's instructions, ligated,and transformed into Bacillus subtilis 168Δ4 competent cells.Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml after incubation at 30°C. for 24-48 hours. Transformants carrying the correct plasmid wereidentified on a 0.8% agarose-0.5X TBE gel by restriction analysis withSspl. The resulting construct was designated pMRT122 (FIG. 37).

[0308] Plasmid pMRT122 was transformed into Bacillus subtilis A164Δ5competent cells. Transformants were selected on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 μg of lincomycin per mlafter incubation at 30° C. for 24-48 hours. The plasmid was introducedinto the chromosome of Bacillus subtilis A164Δ5 via homologousrecombination into the cypX locus by incubating a freshly streaked plateof Bacillus subtilis A164Δ5 (pMRT086) cells at 45° C. for 16 hours andselecting for healthy growing colonies. Genomic DNA was isolated fromthis strain using a QIAGEN tip-20 column according to the manufacturer'sinstructions and used to transform Bacillus subtilis RB187 (Example 9).Transformants were selected on TBAB plates supplemented with 1 μg oferythromycin and 25 μg of lincomycin per ml after incubation at 45° C.for 16 hours. At this temperature, the pE194 replicon is unable toreplicate. Cells are able to maintain erythromycin resistance only bymaintaining the plasmid in the bacterial chromosome.

[0309] The plasmid was removed from the chromosome via homologousrecombination resulting in the deletion of a portion of the cypX gene onthe chromosome by growing the transformants in Luria-Bertani (LB) mediumwithout selection at the permissive temperature of 34° C. for manygenerations. At this temperature the pE194 origin of replication isactive and actually promotes the excision of the plasmid from thechromosome (Molecular Biological Methods for Bacillus, edited by C. R.Harwood and S. M. Cutting, 1990, John Wiley and Sons Ltd.).

[0310] After several generations of outgrowth the cells were plated onnon-selective LB agar plates and colonies which had lost the plasmid andwere now cypX-deleted and producing hyaluronic acid were identified asfollows: (1) cell patches were “wet” when plated on minimal platesindicating production of hyaluronic acid, (2) erythromycin sensitivityindicated loss of the pE194-based plasmid, and (3) PCR confirmed thepresence of the 800 bp cypX deletion in the strain of interest by usingprimers 34 and 45.

[0311] Chromosomal DNA from potential cypX deletants was isolated usingthe REDextract-N-Amp™ Plant PCR kits as follows: Single Bacilluscolonies were inoculated into 100 μl of Extraction Solution, incubatedat 95° C. for 10 minutes, and then diluted with an equal volume ofDilution Solution. PCR was performed using 4 μl of extracted DNA inconjunction with the REDextract-N-Amp™ PCR Reaction Mix and the desiredprimers according to the manufacturer's instructions, using PCR cyclingconditions as described in Example 5. PCR reaction products werevisualized using a 0.8% agarose-0.5X TBE gel. The verified strain wasdesignated Bacillus subtilis RB197.

Example 13: Construction of Bacillus subtilis RB200

[0312] The cypX gene of Bacillus subtilis RB192 was deleted using thesame methods described in Example 9 for Bacillus subtilis RB187. Theresultant strain was designated Bacillus subtilis RB200.

Example 14: Construction of Bacillus subtilis RB202

[0313] Bacillus subtilis A164Δ5ΔcypX was constructed as follows: PlasmidpMRT122 (Example 12) was transformed into Bacillus subtilis A164Δ5competent cells. Transformants were selected on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 μg of lincomycin per mlafter incubation at 30° C. for 24-48 hours. The plasmid was introducedinto the chromosome of Bacillus subtilis A164Δ5 via homologousrecombination into the cypX locus by incubating a freshly streaked plateof Bacillus subtilis A164Δ5 (pMRT086) cells at 45° C. for 16 hours andselecting for healthy growing colonies. The plasmid was removed from thechromosome via homologous recombination resulting in the deletion of aportion of the cypX gene on the chromosome by growing the transformantsin Luria-Bertani (LB) medium without selection at the permissivetemperature of 34° C. for many generations. At this temperature thepE194 origin of replication is active and actually promotes the excisionof the plasmid from the chromosome (Molecular Biological Methods forBacillus, edited by C. R. Harwood and S. M. Cutting, 1990, John Wileyand Sons Ltd.). After several generations of outgrowth the cells wereplated on non-selective LB agar plates and colonies which had lost theplasmid and were now cypX-deleted were identified as follows: (1)erythromycin sensitivity indicated loss of the pE194-based plasmid, and(2) PCR confirmed the presence of the 800 bp cypX deletion in the strainof interest by using primers 34 and 45 as described above. The verifiedstrain was designated Bacillus subtilis A164□5□cypX.

[0314] Bacillus subtilis A164Δ5ΔcypX was made competent and transformedwith Bacillus subtilis TH1 genomic DNA (Example 7) isolated using aQIAGEN tip-20 column according to the manufacturer's instructions.Transformants were selected on TBAB plates containing 5 μg ofchloramphenicol per ml at 37° C. The Bacillus subtilis A164Δ5ΔcypXhasA/hasB/hasC/hasD integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB201. The cat gene was deleted fromBacillus subtilis RB201 using the same method described in Example 9.The resultant strain was designated Bacillus subtilis RB202.

Example 15: Construction of Bacillus subtilis MF002 (tuaD/gtaB)

[0315] Plasmid pHA3 (Example 2, FIG. 9) was digested with Asp718. Thedigested plasmid was then blunted by first inactivating the restrictionenzyme at 85° C. for 30 minutes. Blunting was performed by adding 0.5 μlof 10 mM each dNTPs, 1 μl of 1 U/μl T4 polymerase and incubating at 11°C. for 10 minutes. Finally the polymerase was inactivated by incubatingthe reaction at 75° C. for 10 minutes. The digested plasmid was thenpurified using a QIAquick DNA Purification Kit according to themanufacturer's instructions and finally digested with Notl. The smallestplasmid fragment of approximately 2522 bp was then gel-purified using aQIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions. The recovered DNA insert(tuaD/gtaB) was then ligated with the vector DNA described below.

[0316] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdigested with Ecl136ll. The digested plasmid was then purified using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with Notl. The largest plasmidfragment of approximately 6800 bp was gel-purified from a 0.8%agarose-0.5X TBE gel using a QIAquick DNA Gel Extraction Kit accordingto the manufacturer's instructions.

[0317] The recovered vector and DNA insert were ligated using the RapidDNA Cloning Kit according to the manufacturer's instructions. Prior totransformation in Bacillus subtilis, the ligation described above waslinearized using Scal to ensure double cross-over integration in thechromosome rather than single cross-over integration in the chromosome.Bacillus subtilis 168Δ4 competent cells were transformed with theligation digested with the restriction enzyme Scal.

[0318] Bacillus subtilis chloramphenicol-resistant transformants wereselected on TBAB plates supplemented with 5 μg of chloramphenicol perml. To screen for integration of the plasmid by double cross-over at theamyE locus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive transformants wereisolated.

[0319] Chromosomal DNA from chloramphenicol resistant and neomycinsensitive Bacillus subtilis 168Δ4 transformants was isolated using theREDextract-N-Amp™ Plant PCR kits (Sigma Chemical Company, St. Louis,Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl ofExtraction Solution, incubated at 95° C. for 10 minutes, and thendiluted with an equal volume of Dilution Solution. PCR was performedusing 4 μl of extracted DNA in conjunction with the REDextract-N-Amp PCRReaction Mix and the desired primers according to the manufacturer'sinstructions, with PCR cycling conditions described in Example 5.

[0320] PCR amplifications were performed on these transformants usingthe synthetic oligonucleotides described below to confirm theabsence/presence and integrity of the genes hasA, gtaB, and tuaD of theoperon of the Bacillus subtilis transformants. Primers 3 and 8 were usedto confirm the absence of the hasA gene, primer 71 and primer 15 toconfirm the presence of the tuaD gene, and primers 20 and 71 to confirmthe presence of the gtaB gene. PCR reaction products were visualized ina 0.8% agarose-0.5X TBE gel. The verified strain, a Bacillus subtilis168Δ4 hasA/tuaD/gtaB integrant, was designated Bacillus subtilis RB176.Primer 71: 5′-AACTATTGCCGATGATAAGC-3′ (binds upstream of tuaD) (SEQ IDNO: 85)

[0321] Genomic DNA was isolated from the chloramphenicol resistant, andneomycin sensitive Bacillus subtilis RB176 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. The Bacillussubtilis RB176 genomic DNA was used to transform competent Bacillussubtilis A164Δ5. Transformants were selected on TBAB plates containing 5μg of chloramphenicol per ml, and grown at 37° C. A Bacillus subtilisA164Δ5 tuaD/gtaB integrant was designated Bacillus subtilis RB177.

[0322] The cat gene was deleted in strain Bacillus subtilis RB177 usingthe method described in Example 9. The resultant strain was designatedBacillus subtilis MF002.

Example 16: Construction of the pel integration plasmid pRB162

[0323] Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdouble-digested with Sacl and Aatll. The largest plasmid fragment ofapproximately 6193 bp was gel-purified using a QIAquick DNA GelExtraction Kit from a 0.8% agarose-0.5X TBE gel according to themanufacturer's instructions. The recovered vector DNA was then ligatedwith the DNA insert described below.

[0324] The 5′ and 3′ fragments of a Bacillus subtilis pectate lyase gene(pel, accession number BG10840, SEQ ID NOs. 86 [DNA sequence] and 87[deduced amino acid sequence]) was PCR amplified from Bacillus subtilis168 (BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) usingprimers 72 (introduces 5′ Spel restriction site) and 73 (introduces 3′Sall restriction site) for the 5′ pel fragment and primers 74(introduces 5′ Sacl/BamHl restriction sites) and 75 (introduces 3′Notl/Aatll restriction sites) for the 3′ pel fragment: Primer 72:5′-ACTAGTAATGATGGCTGGGGCGCGTA-3′ (SEQ ID NO: 88) Primer 73:5′-GTCGACATGTTGTCGTATTGTGAGTT-3′ (SEQ ID NO: 89) Primer 74:5′-GAGCTCTACAACGCTTATGGATCCGCGGCCGCGGCGGCACACACATCTGGAT-3′ (SEQ ID NO:90) Primer 75: 5′-GACGTCAGCCCGTTTGCAGCCGATGC-3′ (SEQ ID NO: 91)

[0325] PCR amplifications were carried out in triplicate in 30 μlreactions composed of 50 ng of Bacillus subtilis 168 chromosomal DNA,0.4 μM each of primer pair 72/73 for the 5′ pel fragment or primer pair74/75 for the 3′ pel fragment, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1X PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 9 minutes; 3 cycleseach at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes. The PCRproducts were visualized using a 0.8% agarose-0.5X TBE gel. The expectedfragments were approximately 530 bp for the 5′ pel fragment and 530 bpfor the 3′ pel fragment.

[0326] The 530 bp 5′ pel and 530 bp 3′ pel PCR fragments were clonedinto pCR2.1 using the TA-TOPO Cloning Kit and transformed into E. coliOneShot™ competent cells according to the manufacturers' instructions.Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml incubated at 37° C. Plasmid DNA from thesetransformants was purified using a QIAGEN robot according to themanufacturer's instructions and the DNA sequence of the insertsconfirmed by DNA sequencing using the primers described above (primers72 and 73 for 5′ pel and primers 74 and 75 for 3′ pel). The plasmidsharboring the 530 bp and the 530 bp PCR fragments were designatedpCR2.1-pel 5′ and pCR2.1-pel3′, respectively (FIGS. 38 and 39,respectively).

[0327] Plasmid pCR2.1-pel3′ was double-digested with Sacl and Aatll. Thesmallest plasmid fragment of approximately 530 bp was gel-purified usinga QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions.

[0328] The recovered vector (pDG268MCSΔneo/scBAN) and DNA insert (3′pel) were ligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. The ligation mix was transformed into E.coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.).Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml at 37° C.

[0329] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby Sacl and Aatll digestion on a 0.8% agarose gel using 0.5X TBE buffer.The correct plasmid was identified by the presence of an approximately530 bp Sacl/Aatll 3′ pel fragment and was designated pRB161 (FIG. 40).

[0330] Plasmid pRB161 was double-digested with Spel and Sall. Thelargest plasmid fragment of approximately 5346 bp was gel-purified usinga QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions. The recovered vector DNAwas then ligated with the DNA insert described below.

[0331] Plasmid pCR2.1-pel5′ was double-digested with Spel and Sall. Thesmallest plasmid fragment of approximately 530 bp was gel-purified usinga QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions.

[0332] The recovered vector (pDG268MCSΔneo/scBAN/pel 3′) and insert (pel5′) DNA were ligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. The ligation mix was transformed into E.coil SURE competent cells (Stratagene, Inc., La Jolla, Calif.).Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml.

[0333] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby Spel and Sall digestion on a 0.8% agarose gel using 0.5X TBE buffer.The correct plasmid was identified by the presence of an approximately530 bp Spel/Sall pel 5′ fragment and was designated pRB162 (FIG. 41).

Example 17: Construction of pRB156

[0334] Plasmid pHA7 (Example 4, FIG. 13) was digested with Hpal. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions and finally digested withAsp718. The double-digested plasmid was then blunted by firstinactivating the restriction enzyme at 85° C. for 30 minutes. Bluntingwas performed by adding 0.5 μl of 10 mM each dNTPs and 1 μl of 1 U/μl ofT4 polymerase and incubating at 11° C. for 10 minutes. The polymerasewas then inactivated by incubating the reaction at 75° C. for 10minutes. The largest plasmid fragment of approximately 8600 bp was thengel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5X TBE gel according to the manufacturer's instructions. Therecovered DNA insert (pDG268Δneo-crylllAstab/sehasA) was then re-ligatedusing the Rapid DNA Cloning Kit according to the manufacturer'sinstructions.

[0335] The ligation mix was transformed into E. coli SURE competentcells (Stratagene, Inc., La Jolla, Calif.). Transformants were selectedon 2X YT agar plates supplemented with 100 μg of ampicillin per ml at37° C. Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby Scal digestion on a 0.8% agarose gel using 0.5X TBE buffer. Thecorrect plasmid was identified by the presence of an approximately 8,755bp fragment and was designated pRB156 (FIG. 42).

Example 18: Construction of Bacillus subtilis MF009

[0336] The hasA gene under control of the scBAN promoter was introducedinto the pectate lyase gene (pel) locus of Bacillus subtilis MF002 togenerate Bacillus subtilis MF009.

[0337] Plasmid pRB156 was digested with Sacl. The digested plasmid wasthen purified using a QIAquick DNA Purification Kit according to themanufacturer's instructions, and finally digested with Notl. Thesmallest plasmid fragment of approximately 1,377 bp was gel-purifiedusing a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions. The recovered DNA insertwas then ligated with the vector DNA described below.

[0338] Plasmid pRB162 (Example 16, FIG. 41) was digested with Notl. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withSacl. The largest plasmid fragment of approximately 5850 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5X TBE gel according to the manufacturer's instructions. Therecovered vector DNA was then ligated with the DNA insert describedabove.

[0339] The ligation mixture was transformed directly in Bacillussubtilis 168Δ4 competent cells. Bacillus subtilischloramphenicol-resistant transformants were selected on TBAB platessupplemented with 5 μg of chloramphenicol per ml at 37° C. To screen forintegration of the plasmid by double cross-over at the pel locus,Bacillus subtilis primary transformants were patched on TBAB platessupplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml. Integration of theplasmid by double cross-over at the pel locus does not incorporate theneomycin resistance gene and therefore renders the strain neomycinsensitive. Using this plate screen, chloramphenicol resistant andneomycin sensitive transformants were isolated.

[0340] Genomic DNA was isolated from the chloramphenicol resistant andneomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. This genomicDNA was used to transform competent Bacillus subtilis MF002 (Example15). Transformants were selected on TBAB plates containing 5 μg ofchloramphenicol per ml and grown at 37° C. The Bacillus subtilis A164Δ5hasA and tuaD/gtaB integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis MF009.

Example 19: Construction of Bacillus subtilis MF010

[0341] Plasmid pDG268MCSΔneo/BAN/Sav (U.S. Pat. No. 5,955,310) wasdigested with Notl. The digested plasmid was then purified using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with Sfil. The smallest plasmidfragment of approximately 185 bp was gel-purified using a QIAquick DNAGel Extraction Kit from a 0.8% agarose-0.5X TBE gel according to themanufacturer's instructions. The recovered DNA insert was then ligatedwith the vector DNA described below.

[0342] Plasmid pRB162 (Example 16, FIG. 41) was digested with Notl. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withSfil. The largest plasmid fragment of approximately 5747 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5X TBE gel according to the manufacturer's instructions. Therecovered vector DNA was then ligated with the DNA insert describedabove.

[0343] The recovered vector and DNA insert were ligated using the RapidDNA Cloning Kit according to the manufacturer's instructions. Theligation mix was transformed into E. coli XLI Blue competent cells.Transformants were selected on 2X YT agar plates supplemented with 100μg of ampicillin per ml.

[0344] Plasmid DNA was purified from several transformants using aQIAGEN robot according to the manufacturer's instructions and analyzedby BamHl digestion on a 0.8% agarose gel using 0.5X TBE buffer. Thecorrect plasmid was identified by the linearization of the plasmid whichprovides an approximately 7,156 bp fragment and was designated pRB164(FIG. 43).

[0345] Plasmid pRB156 (Example 17, FIG. 42) was digested with Sacl. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withNotl. The smallest plasmid fragment of approximately 1377 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5X TBE gel according to the manufacturer's instructions. Therecovered DNA insert was then ligated with the vector DNA describedbelow.

[0346] Plasmid pRB164 was digested with Notl. The digested plasmid wasthen purified using a QIAquick DNA Purification Kit according to themanufacturer's instructions, and finally digested with Sacl. The largestplasmid fragment of approximately 5922 bp was gel-purified using aQIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5X TBE gelaccording to the manufacturer's instructions. The recovered vector DNAwas then ligated with the DNA insert described above.

[0347] This ligation mix was transformed directly in Bacillus subtilis168Δ4 competent cells. Bacillus subtilis chloramphenicol-resistanttransformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. To screen for integration of theplasmid by double cross-over at the amyE locus, Bacillus subtilisprimary transformants were patched on TBAB plates supplemented with 6 μgof neomycin per ml and on TBAB plates supplemented with 5 μg ofchloramphenicol per ml. Integration of the plasmid by double cross-overat the amyE locus does not incorporate the neomycin resistance gene andtherefore renders the strain neomycin sensitive. Using this platescreen, chloramphenicol resistant and neomycin sensitive transformantswere isolated.

[0348] Genomic DNA was isolated from the chloramphenicol resistant andneomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. This genomicDNA was used to transform competent Bacillus subtilis MF002 (Example15). Transformants were selected on minimal plates containing 5 μg ofchloramphenicol per ml and grown at 37° C. for 16 hours. A Bacillussubtilis A164Δ5 BAN/hasA and scBAN/tuaD/gtaB integrant was identified byits “wet” phenotype and designated Bacillus subtilis MF010.

Example 20: Fermentations

[0349] The ability of the Bacillus subtilis strains listed in Table 1 toproduce hyaluronic acid was evaluated under various growth conditions.TABLE 1 B. subtilis Strain promoter/gene complement catΔ cypXΔ RB161scBAN/hasA/tuaD/gtaB no no RB163 scBAN/hasA/tuaD/gcaD no no TH-1scBANhasA/hasB/hasC/hasD no no RB184 scBAN/hasA/tuaD no no RB187scBAN/hasA/tuaD/gtaB yes no RB192 scBAN/hasA/tuaD yes no RB194scBAN/hasA/tuaD/gtaB yes yes RB197 scBAN/hasA/tuaD/gtaB yes yes RB200scBAN/hasA/tuaD yes yes RB202 scBAN/hasA/hasB/hasC/hasD yes yes MF009scBAN/tuaD/gtaB no no scBAN/hasA MF010 scBAN/tuaD/gtaB no no BAN/hasA

[0350] The Bacillus subtilis strains were fermented in standard smallfermenters in a medium composed per liter of 6.5 g of KH₂PO₄, 4.5 g ofNa₂HPO₄, 3.0 g of (NH₄)₂SO₄, 2.0 g of Na₃-citrate-2H₂O, 3.0 g ofMgSO₄.7H₂O, 6.0 ml of Mikrosoy-2, 0.15 mg of biotin (1 ml of 0.15 mg/mlethanol), 15.0 g of sucrose, 1.0 ml of SB 2066, 2.0 ml of P2000, 0.5 gof CaCl₂.2H₂O. The medium was pH 6.3 to 6.4 (unadjusted) prior toautoclaving. The CaCl₂.2H₂O was added after autoclaving.

[0351] The seed medium used was B-3, i.e., Agar-3 without agar, or“S/S-1” medium. The Agar-3 medium was composed per liter of 4.0 g ofnutrient broth, 7.5 g of hydrolyzed protein, 3.0 g of yeast extract, 1.0g of glucose, and 2% agar. The pH was not adjusted; pH beforeautoclaving was approximately 6.8; after autoclaving approximately pH7.7.

[0352] The sucrose/soy seed flask medium (S/S-1) was composed per literof 65 g of sucrose, 35 g of soy flour, 2 g of Na₃-citrate-.2H₂O, 4 g ofKH₂PO₄, 5 g of Na₂HPO₄, and 6 ml of trace elements. The medium wasadjusted pH to about 7 with NaOH; after dispensing the medium to flasks,0.2% vegetable oil was added to suppress foaming. Trace elements wascomposed per liter of 100 g of citric acid-H₂O, 20 g of FeSO₄.7H₂O, 5 gof MnSO₄.H₂O, 2 g of CuSO₄.5H₂O, and 2 g of ZnCl₂.

[0353] The pH was adjusted to 6.8-7.0 with ammonia before inoculation,and controlled thereafter at pH 7.0±0.2 with ammonia and H₃PO₄. Thetemperature was maintained at 37° C. Agitation was at a maximum of 1300RPM using two 6-bladed rushton impellers of 6 cm diameter in 3 litertank with initial volume of 1.5 liters. The aeration had a maximum of1.5 VVM.

[0354] For feed, a simple sucrose solution was used. Feed started atabout 4 hours after inoculation, when dissolved oxygen (D.O.) was stillbeing driven down (i.e., before sucrose depletion). The feed rate wasramped linearly from 0 to approximately 6 g sucrose/L₀-hr over a 7 hourtime span. A lower feed rate, ramped linearly from 0 to approximately 2g sucrose/L₀-hr, was also used in some fermentations.

[0355] Viscosity was noticeable by about 10 hours and by 24 hoursviscosity was very high, causing the D.O. to bottom-out. End-pointviscosity reached 3,220 cP. Cell mass development reached a near maximum(12 to 15 g/liter) by 20 hours. Cells were removed by diluting 1 partculture with 3 parts water, mixing well and centrifuging at about 30,000x g to produce a clear supernatant and cell pellet, which can be washedand dried.

[0356] Assays of hyaluronic acid concentration were performed using theELISA method, based on a hyaluronan binding protein (protein and kitscommercially available from Seikagaku America, Falmouth, Mass.).

[0357] Bacillus subtilis RB 161 and RB163 were cultured in batch andfed-batch fermentations. In the fed-batch processes, the feed rate wasvaried between cultures of Bacillus subtilis strains RB163 and RB161.Assays of hyaluronic acid concentrations were again performed using theELISA method. The results are provided in Table 2. TABLE 2 HA (relativeStrain and Growth yield) Conditions ELISA method RB-161 0.7 ± 0.1(hasA/tuaD/gtaB) simple batch RB-163 0.9 ± 0.1 (hasA/tuaD/gcaD) fedbatch˜6 g sucrose/L₀-hr RB161 0.9 ± 0.1 (hasA/tuaD/gtaB) fed batch˜6 gsucrose/L₀-hr RB-163 1.0 ± 0..2 (hasA/tuaD/gcaD) fed batch˜2 gsucrose/L₀-hr RB161 1.0 ± 0..1 (hasA/tuaD/gtaB) fed batch˜2 gsucrose/L₀-hr

[0358] The results of the culture assays for the same strain at a fedbatch rate of 2 g/L sucrose/L₀-hr compared to 6 g/L sucrose/L₀-hrdemonstrated that a faster sucrose feed rate did not significantlyimprove titers.

[0359] A summary of the Bacillus strains run under same conditions (fedbatch at approximately 2 g sucrose/L₀-hr, 37° C.) is shown in FIG. 44.In FIG. 44, ± values indicate standard deviation of data from multipleruns under the same conditions. Data without ± values are from singleruns. Hyaluronic acid concentrations were determined using the modifiedcarbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).

[0360] A summary of peak hyaluronic acid weight average molecularweights (MDa) obtained from fermentation of the recombinant Bacillussubtilis strains under the same conditions (fed batch at approximately 2g sucrose/L₀-hr, 37° C.) is shown in FIG. 45. Molecular weights weredetermined using a GPC MALLS assay. Data was gathered from GPC MALLSassays using the following procedure. GPC-MALLS (gel permeation orsize-exclusion) chromatography coupled with multi-angle laser lightscattering) is widely used to characterize high molecular weight (MW)polymers. Separation of polymers is achieved by GPC, based on thedifferential partitioning of molecules of different MW between eluentand resin. The average molecular weight of an individual polymer isdetermined by MALLS based the differential scattering extent/angle ofmolecules of different MW. Principles of GPC-MALLS and protocols suitedfor hyaluronic acid are described by Ueno et al., 1988, Chem. Pharm.Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and WyattTechnologies, 1999, “Light Scattering University DAWN Course Manual” and“DAWN EOS Manual” Wyatt Technology Corporation, Santa Barbara, Calif.).An Agilent 1100 isocratic HPLC, a Tosoh Biosep G6000 PWxl column for theGPC, and a Wyatt Down EOS for the MALLS were used. An Agilent G1362Arefractive index detector was linked downstream from the MALLS foreluate concentration determination. Various commercial hyaluronic acidproducts with known molecular weights served as standards.

Deposit of Biological Material

[0361] The following biological material has been deposited under theterms of the Budapest Treaty with the Agricultural Research Service Pat.Culture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:Deposit Accession Number Date of Deposit E. coli XL10 Gold kan (pMRT106)NRRL B-30536 Dec. 12, 2001

[0362] The strain has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0363] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0364] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1 108 1 1251 DNA Streptococcus equisimilis CDS (1)..(1251) 1 atg aga acatta aaa aac ctc ata act gtt gtg gcc ttt agt att ttt 48 Met Arg Thr LeuLys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe 1 5 10 15 tgg gta ctgttg att tac gtc aat gtt tat ctc ttt ggt gct aaa gga 96 Trp Val Leu LeuIle Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly 20 25 30 agc ttg tca atttat ggc ttt ttg ctg ata gct tac cta tta gtc aaa 144 Ser Leu Ser Ile TyrGly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys 35 40 45 atg tcc tta tcc tttttt tac aag cca ttt aag gga agg gct ggg caa 192 Met Ser Leu Ser Phe PheTyr Lys Pro Phe Lys Gly Arg Ala Gly Gln 50 55 60 tat aag gtt gca gcc attatt ccc tct tat aac gaa gat gct gag tca 240 Tyr Lys Val Ala Ala Ile IlePro Ser Tyr Asn Glu Asp Ala Glu Ser 65 70 75 80 ttg cta gag acc tta aaaagt gtt cag cag caa acc tat ccc cta gca 288 Leu Leu Glu Thr Leu Lys SerVal Gln Gln Gln Thr Tyr Pro Leu Ala 85 90 95 gaa att tat gtt gtt gac gatgga agt gct gat gag aca ggt att aag 336 Glu Ile Tyr Val Val Asp Asp GlySer Ala Asp Glu Thr Gly Ile Lys 100 105 110 cgc att gaa gac tat gtg cgtgac act ggt gac cta tca agc aat gtc 384 Arg Ile Glu Asp Tyr Val Arg AspThr Gly Asp Leu Ser Ser Asn Val 115 120 125 att gtt cac cgg tca gaa aaaaat caa gga aag cgt cat gca cag gcc 432 Ile Val His Arg Ser Glu Lys AsnGln Gly Lys Arg His Ala Gln Ala 130 135 140 tgg gcc ttt gaa aga tca gacgct gat gtc ttt ttg acc gtt gac tca 480 Trp Ala Phe Glu Arg Ser Asp AlaAsp Val Phe Leu Thr Val Asp Ser 145 150 155 160 gat act tat atc tac cctgat gct tta gag gag ttg tta aaa acc ttt 528 Asp Thr Tyr Ile Tyr Pro AspAla Leu Glu Glu Leu Leu Lys Thr Phe 165 170 175 aat gac cca act gtt tttgct gcg acg ggt cac ctt aat gtc aga aat 576 Asn Asp Pro Thr Val Phe AlaAla Thr Gly His Leu Asn Val Arg Asn 180 185 190 aga caa acc aat ctc ttaaca cgc ttg aca gat att cgc tat gat aat 624 Arg Gln Thr Asn Leu Leu ThrArg Leu Thr Asp Ile Arg Tyr Asp Asn 195 200 205 gct ttt ggc gtt gaa cgagct gcc caa tcc gtt aca ggt aat att ctc 672 Ala Phe Gly Val Glu Arg AlaAla Gln Ser Val Thr Gly Asn Ile Leu 210 215 220 gtt tgc tca ggc ccg cttagc gtt tac aga cgc gag gtg gtt gtt cct 720 Val Cys Ser Gly Pro Leu SerVal Tyr Arg Arg Glu Val Val Val Pro 225 230 235 240 aac ata gat aga tacatc aac cag acc ttc ctg ggt att cct gta agt 768 Asn Ile Asp Arg Tyr IleAsn Gln Thr Phe Leu Gly Ile Pro Val Ser 245 250 255 atc ggt gat gac aggtgc ttg acc aac tat gca act gat tta gga aag 816 Ile Gly Asp Asp Arg CysLeu Thr Asn Tyr Ala Thr Asp Leu Gly Lys 260 265 270 act gtt tat caa tccact gct aaa tgt att aca gat gtt cct gac aag 864 Thr Val Tyr Gln Ser ThrAla Lys Cys Ile Thr Asp Val Pro Asp Lys 275 280 285 atg tct act tac ttgaag cag caa aac cgc tgg aac aag tcc ttc ttt 912 Met Ser Thr Tyr Leu LysGln Gln Asn Arg Trp Asn Lys Ser Phe Phe 290 295 300 aga gag tcc att atttct gtt aag aaa atc atg aac aat cct ttt gta 960 Arg Glu Ser Ile Ile SerVal Lys Lys Ile Met Asn Asn Pro Phe Val 305 310 315 320 gcc cta tgg accata ctt gag gtg tct atg ttt atg atg ctt gtt tat 1008 Ala Leu Trp Thr IleLeu Glu Val Ser Met Phe Met Met Leu Val Tyr 325 330 335 tct gtg gtg gatttc ttt gta gac aat gtc aga gaa ttt gat tgg ctc 1056 Ser Val Val Asp PhePhe Val Asp Asn Val Arg Glu Phe Asp Trp Leu 340 345 350 agg gtt ttg gccttt ctg gtg att atc ttc att gtt gct ctt tgt cgt 1104 Arg Val Leu Ala PheLeu Val Ile Ile Phe Ile Val Ala Leu Cys Arg 355 360 365 aat att cac tatatg ctt aag cac ccg ctg tcc ttc ttg tta tct ccg 1152 Asn Ile His Tyr MetLeu Lys His Pro Leu Ser Phe Leu Leu Ser Pro 370 375 380 ttt tat ggg gtactg cat ttg ttt gtc cta cag ccc ttg aaa ttg tat 1200 Phe Tyr Gly Val LeuHis Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr 385 390 395 400 tct ctt tttact att aga aat gct gac tgg gga aca cgt aaa aaa tta 1248 Ser Leu Phe ThrIle Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu 405 410 415 tta 1251 Leu2 417 PRT Streptococcus equisimilis 2 Met Arg Thr Leu Lys Asn Leu IleThr Val Val Ala Phe Ser Ile Phe 1 5 10 15 Trp Val Leu Leu Ile Tyr ValAsn Val Tyr Leu Phe Gly Ala Lys Gly 20 25 30 Ser Leu Ser Ile Tyr Gly PheLeu Leu Ile Ala Tyr Leu Leu Val Lys 35 40 45 Met Ser Leu Ser Phe Phe TyrLys Pro Phe Lys Gly Arg Ala Gly Gln 50 55 60 Tyr Lys Val Ala Ala Ile IlePro Ser Tyr Asn Glu Asp Ala Glu Ser 65 70 75 80 Leu Leu Glu Thr Leu LysSer Val Gln Gln Gln Thr Tyr Pro Leu Ala 85 90 95 Glu Ile Tyr Val Val AspAsp Gly Ser Ala Asp Glu Thr Gly Ile Lys 100 105 110 Arg Ile Glu Asp TyrVal Arg Asp Thr Gly Asp Leu Ser Ser Asn Val 115 120 125 Ile Val His ArgSer Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala 130 135 140 Trp Ala PheGlu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser 145 150 155 160 AspThr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe 165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn 180 185190 Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn 195200 205 Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220 Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val ValPro 225 230 235 240 Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly IlePro Val Ser 245 250 255 Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala ThrAsp Leu Gly Lys 260 265 270 Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile ThrAsp Val Pro Asp Lys 275 280 285 Met Ser Thr Tyr Leu Lys Gln Gln Asn ArgTrp Asn Lys Ser Phe Phe 290 295 300 Arg Glu Ser Ile Ile Ser Val Lys LysIle Met Asn Asn Pro Phe Val 305 310 315 320 Ala Leu Trp Thr Ile Leu GluVal Ser Met Phe Met Met Leu Val Tyr 325 330 335 Ser Val Val Asp Phe PheVal Asp Asn Val Arg Glu Phe Asp Trp Leu 340 345 350 Arg Val Leu Ala PheLeu Val Ile Ile Phe Ile Val Ala Leu Cys Arg 355 360 365 Asn Ile His TyrMet Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro 370 375 380 Phe Tyr GlyVal Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr 385 390 395 400 SerLeu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu 405 410 415Leu 3 49 DNA Streptococcus equisimilis 3 gagctctata aaaatgaggagggaaccgaa tgagaacatt aaaaaacct 49 4 48 DNA Streptococcus equisimilis 4gttaacgaat tcagctatgt aggtacctta taataatttt ttacgtgt 48 5 20 DNAStreptococcus equisimilis 5 gttgacgatg gaagtgctga 20 6 20 DNAStreptococcus equisimilis 6 atccgttaca ggtaatatcc 20 7 20 DNAStreptococcus equisimilis 7 tccttttgta gccctatgga 20 8 20 DNAStreptococcus equisimilis 8 tcagcacttc catcgtcaac 20 9 20 DNAStreptococcus equisimilis 9 ggatattacc tgtaacggat 20 10 20 DNAStreptococcus equisimilis 10 tccatagggc tacaaaagga 20 11 1383 DNABacillus subtilis CDS (1)..(1383) 11 gtg aaa aaa ata gct gtc att gga acaggt tat gta gga ctc gta tca 48 Val Lys Lys Ile Ala Val Ile Gly Thr GlyTyr Val Gly Leu Val Ser 1 5 10 15 ggc act tgc ttt gcg gag atc ggc aataaa gtt gtt tgc tgt gat atc 96 Gly Thr Cys Phe Ala Glu Ile Gly Asn LysVal Val Cys Cys Asp Ile 20 25 30 gat gaa tca aaa atc aga agc ctg aaa aatggg gta atc cca atc tat 144 Asp Glu Ser Lys Ile Arg Ser Leu Lys Asn GlyVal Ile Pro Ile Tyr 35 40 45 gaa cca ggg ctt gca gac tta gtt gaa aaa aatgtg ctg gat cag cgc 192 Glu Pro Gly Leu Ala Asp Leu Val Glu Lys Asn ValLeu Asp Gln Arg 50 55 60 ctg acc ttt acg aac gat atc ccg tct gcc att cgggcc tca gat att 240 Leu Thr Phe Thr Asn Asp Ile Pro Ser Ala Ile Arg AlaSer Asp Ile 65 70 75 80 att tat att gca gtc gga acg cct atg tcc aaa acaggt gaa gct gat 288 Ile Tyr Ile Ala Val Gly Thr Pro Met Ser Lys Thr GlyGlu Ala Asp 85 90 95 tta acg tac gtc aaa gcg gcg gcg aaa aca atc ggt gagcat ctt aac 336 Leu Thr Tyr Val Lys Ala Ala Ala Lys Thr Ile Gly Glu HisLeu Asn 100 105 110 ggc tac aaa gtg atc gta aat aaa agc aca gtc ccg gttgga aca ggg 384 Gly Tyr Lys Val Ile Val Asn Lys Ser Thr Val Pro Val GlyThr Gly 115 120 125 aaa ctg gtg caa tct atc gtt caa aaa gcc tca aag gggaga tac tca 432 Lys Leu Val Gln Ser Ile Val Gln Lys Ala Ser Lys Gly ArgTyr Ser 130 135 140 ttt gat gtt gta tct aac cct gaa ttc ctt cgg gaa gggtca gcg att 480 Phe Asp Val Val Ser Asn Pro Glu Phe Leu Arg Glu Gly SerAla Ile 145 150 155 160 cat gac acg atg aat atg gag cgt gcc gtg att ggttca aca agt cat 528 His Asp Thr Met Asn Met Glu Arg Ala Val Ile Gly SerThr Ser His 165 170 175 aaa gcc gct gcc atc att gag gaa ctt cat cag ccattc cat gct cct 576 Lys Ala Ala Ala Ile Ile Glu Glu Leu His Gln Pro PheHis Ala Pro 180 185 190 gtc att aaa aca aac cta gaa agt gca gaa atg attaaa tac gcc gcg 624 Val Ile Lys Thr Asn Leu Glu Ser Ala Glu Met Ile LysTyr Ala Ala 195 200 205 aat gca ttt ctg gcg aca aag att tcc ttt atc aacgat atc gca aac 672 Asn Ala Phe Leu Ala Thr Lys Ile Ser Phe Ile Asn AspIle Ala Asn 210 215 220 att tgt gag cga gtc ggc gca gac gtt tca aaa gttgct gat ggt gtt 720 Ile Cys Glu Arg Val Gly Ala Asp Val Ser Lys Val AlaAsp Gly Val 225 230 235 240 ggt ctt gac agc cgt atc ggc aga aag ttc cttaaa gct ggt att gga 768 Gly Leu Asp Ser Arg Ile Gly Arg Lys Phe Leu LysAla Gly Ile Gly 245 250 255 ttc ggc ggt tca tgt ttt cca aag gat aca accgcg ctg ctt caa atc 816 Phe Gly Gly Ser Cys Phe Pro Lys Asp Thr Thr AlaLeu Leu Gln Ile 260 265 270 gca aaa tcg gca ggc tat cca ttc aag ctc atcgaa gct gtc att gaa 864 Ala Lys Ser Ala Gly Tyr Pro Phe Lys Leu Ile GluAla Val Ile Glu 275 280 285 acg aac gaa aag cag cgt gtt cat att gta gataaa ctt ttg act gtt 912 Thr Asn Glu Lys Gln Arg Val His Ile Val Asp LysLeu Leu Thr Val 290 295 300 atg gga agc gtc aaa ggg aga acc att tca gtcctg gga tta gcc ttc 960 Met Gly Ser Val Lys Gly Arg Thr Ile Ser Val LeuGly Leu Ala Phe 305 310 315 320 aaa ccg aat acg aac gat gtg aga tcc gctcca gcg ctt gat att atc 1008 Lys Pro Asn Thr Asn Asp Val Arg Ser Ala ProAla Leu Asp Ile Ile 325 330 335 cca atg ctg cag cag ctg ggc gcc cat gtaaaa gca tac gat ccg att 1056 Pro Met Leu Gln Gln Leu Gly Ala His Val LysAla Tyr Asp Pro Ile 340 345 350 gct att cct gaa gct tca gcg atc ctt ggcgaa cag gtc gag tat tac 1104 Ala Ile Pro Glu Ala Ser Ala Ile Leu Gly GluGln Val Glu Tyr Tyr 355 360 365 aca gat gtg tat gct gcg atg gaa gac actgat gca tgc ctg att tta 1152 Thr Asp Val Tyr Ala Ala Met Glu Asp Thr AspAla Cys Leu Ile Leu 370 375 380 acg gat tgg ccg gaa gtg aaa gaa atg gagctt gta aaa gtg aaa acc 1200 Thr Asp Trp Pro Glu Val Lys Glu Met Glu LeuVal Lys Val Lys Thr 385 390 395 400 ctc tta aaa cag cca gtc atc att gacggc aga aat tta ttt tca ctt 1248 Leu Leu Lys Gln Pro Val Ile Ile Asp GlyArg Asn Leu Phe Ser Leu 405 410 415 gaa gag atg cag gca gcc gga tac atttat cac tct atc ggc cgt ccc 1296 Glu Glu Met Gln Ala Ala Gly Tyr Ile TyrHis Ser Ile Gly Arg Pro 420 425 430 gct gtt cgg gga acg gaa ccc tct gacaag tat ttt ccg ggc ttg ccg 1344 Ala Val Arg Gly Thr Glu Pro Ser Asp LysTyr Phe Pro Gly Leu Pro 435 440 445 ctt gaa gaa ttg gct aaa gac ttg ggaagc gtc aat tta 1383 Leu Glu Glu Leu Ala Lys Asp Leu Gly Ser Val Asn Leu450 455 460 12 461 PRT Bacillus subtilis 12 Val Lys Lys Ile Ala Val IleGly Thr Gly Tyr Val Gly Leu Val Ser 1 5 10 15 Gly Thr Cys Phe Ala GluIle Gly Asn Lys Val Val Cys Cys Asp Ile 20 25 30 Asp Glu Ser Lys Ile ArgSer Leu Lys Asn Gly Val Ile Pro Ile Tyr 35 40 45 Glu Pro Gly Leu Ala AspLeu Val Glu Lys Asn Val Leu Asp Gln Arg 50 55 60 Leu Thr Phe Thr Asn AspIle Pro Ser Ala Ile Arg Ala Ser Asp Ile 65 70 75 80 Ile Tyr Ile Ala ValGly Thr Pro Met Ser Lys Thr Gly Glu Ala Asp 85 90 95 Leu Thr Tyr Val LysAla Ala Ala Lys Thr Ile Gly Glu His Leu Asn 100 105 110 Gly Tyr Lys ValIle Val Asn Lys Ser Thr Val Pro Val Gly Thr Gly 115 120 125 Lys Leu ValGln Ser Ile Val Gln Lys Ala Ser Lys Gly Arg Tyr Ser 130 135 140 Phe AspVal Val Ser Asn Pro Glu Phe Leu Arg Glu Gly Ser Ala Ile 145 150 155 160His Asp Thr Met Asn Met Glu Arg Ala Val Ile Gly Ser Thr Ser His 165 170175 Lys Ala Ala Ala Ile Ile Glu Glu Leu His Gln Pro Phe His Ala Pro 180185 190 Val Ile Lys Thr Asn Leu Glu Ser Ala Glu Met Ile Lys Tyr Ala Ala195 200 205 Asn Ala Phe Leu Ala Thr Lys Ile Ser Phe Ile Asn Asp Ile AlaAsn 210 215 220 Ile Cys Glu Arg Val Gly Ala Asp Val Ser Lys Val Ala AspGly Val 225 230 235 240 Gly Leu Asp Ser Arg Ile Gly Arg Lys Phe Leu LysAla Gly Ile Gly 245 250 255 Phe Gly Gly Ser Cys Phe Pro Lys Asp Thr ThrAla Leu Leu Gln Ile 260 265 270 Ala Lys Ser Ala Gly Tyr Pro Phe Lys LeuIle Glu Ala Val Ile Glu 275 280 285 Thr Asn Glu Lys Gln Arg Val His IleVal Asp Lys Leu Leu Thr Val 290 295 300 Met Gly Ser Val Lys Gly Arg ThrIle Ser Val Leu Gly Leu Ala Phe 305 310 315 320 Lys Pro Asn Thr Asn AspVal Arg Ser Ala Pro Ala Leu Asp Ile Ile 325 330 335 Pro Met Leu Gln GlnLeu Gly Ala His Val Lys Ala Tyr Asp Pro Ile 340 345 350 Ala Ile Pro GluAla Ser Ala Ile Leu Gly Glu Gln Val Glu Tyr Tyr 355 360 365 Thr Asp ValTyr Ala Ala Met Glu Asp Thr Asp Ala Cys Leu Ile Leu 370 375 380 Thr AspTrp Pro Glu Val Lys Glu Met Glu Leu Val Lys Val Lys Thr 385 390 395 400Leu Leu Lys Gln Pro Val Ile Ile Asp Gly Arg Asn Leu Phe Ser Leu 405 410415 Glu Glu Met Gln Ala Ala Gly Tyr Ile Tyr His Ser Ile Gly Arg Pro 420425 430 Ala Val Arg Gly Thr Glu Pro Ser Asp Lys Tyr Phe Pro Gly Leu Pro435 440 445 Leu Glu Glu Leu Ala Lys Asp Leu Gly Ser Val Asn Leu 450 455460 13 26 DNA Bacillus subtilis 13 ggtaccgaca ctgcgaccat tataaa 26 14 49DNA Bacillus subtilis 14 gttaacgaat tccagctatg tatctagaca gcttcaaccaagtaacact 49 15 20 DNA Bacillus subtilis 15 agcatcttaa cggctacaaa 20 1620 DNA Bacillus subtilis 16 tgtgagcgag tcggcgcaga 20 17 20 DNA Bacillussubtilis 17 gggcgcccat gtaaaagcat 20 18 20 DNA Bacillus subtilis 18tttgtagccg ttaagatgct 20 19 20 DNA Bacillus subtilis 19 tctgcgccgactcgctcaca 20 20 20 DNA Bacillus subtilis 20 atgcttttac atgggcgccc 20 21876 DNA Bacillus subtilis CDS (1)..(876) 21 atg aaa aaa gta cgt aaa gccata att cca gca gca ggc tta gga aca 48 Met Lys Lys Val Arg Lys Ala IleIle Pro Ala Ala Gly Leu Gly Thr 1 5 10 15 cgt ttt ctt ccg gct acg aaagca atg ccg aaa gaa atg ctt cct atc 96 Arg Phe Leu Pro Ala Thr Lys AlaMet Pro Lys Glu Met Leu Pro Ile 20 25 30 gtt gat aaa cct acc att caa tacata att gaa gaa gct gtt gaa gcc 144 Val Asp Lys Pro Thr Ile Gln Tyr IleIle Glu Glu Ala Val Glu Ala 35 40 45 ggt att gaa gat att att atc gta acagga aaa agc aag cgt gcg att 192 Gly Ile Glu Asp Ile Ile Ile Val Thr GlyLys Ser Lys Arg Ala Ile 50 55 60 gag gat cat ttt gat tac tct cct gag cttgaa aga aac cta gaa gaa 240 Glu Asp His Phe Asp Tyr Ser Pro Glu Leu GluArg Asn Leu Glu Glu 65 70 75 80 aaa gga aaa act gag ctg ctt gaa aaa gtgaaa aag gct tct aac ctg 288 Lys Gly Lys Thr Glu Leu Leu Glu Lys Val LysLys Ala Ser Asn Leu 85 90 95 gct gac att cac tat atc cgc caa aaa gaa cctaaa ggt ctc gga cat 336 Ala Asp Ile His Tyr Ile Arg Gln Lys Glu Pro LysGly Leu Gly His 100 105 110 gct gtc tgg tgc gca cgc aac ttt atc ggc gatgag ccg ttt gcg gta 384 Ala Val Trp Cys Ala Arg Asn Phe Ile Gly Asp GluPro Phe Ala Val 115 120 125 ctg ctt ggt gac gat att gtt cag gct gaa actcca ggg ttg cgc caa 432 Leu Leu Gly Asp Asp Ile Val Gln Ala Glu Thr ProGly Leu Arg Gln 130 135 140 tta atg gat gaa tat gaa aaa aca ctt tct tctatt atc ggt gtt cag 480 Leu Met Asp Glu Tyr Glu Lys Thr Leu Ser Ser IleIle Gly Val Gln 145 150 155 160 cag gtg ccc gaa gaa gaa aca cac cgc tacggc att att gac ccg ctg 528 Gln Val Pro Glu Glu Glu Thr His Arg Tyr GlyIle Ile Asp Pro Leu 165 170 175 aca agt gaa ggc cgc cgt tat cag gtg aaaaac ttc gtt gaa aaa ccg 576 Thr Ser Glu Gly Arg Arg Tyr Gln Val Lys AsnPhe Val Glu Lys Pro 180 185 190 cct aaa ggc aca gca cct tct aat ctt gccatc tta ggc cgt tac gta 624 Pro Lys Gly Thr Ala Pro Ser Asn Leu Ala IleLeu Gly Arg Tyr Val 195 200 205 ttc acg cct gag atc ttc atg tat tta gaagag cag cag gtt ggc gcc 672 Phe Thr Pro Glu Ile Phe Met Tyr Leu Glu GluGln Gln Val Gly Ala 210 215 220 ggc gga gaa att cag ctc aca gac gcc attcaa aag ctg aat gaa att 720 Gly Gly Glu Ile Gln Leu Thr Asp Ala Ile GlnLys Leu Asn Glu Ile 225 230 235 240 caa aga gtg ttt gct tac gat ttt gaaggc aag cgt tat gat gtt ggt 768 Gln Arg Val Phe Ala Tyr Asp Phe Glu GlyLys Arg Tyr Asp Val Gly 245 250 255 gaa aag ctc ggc ttt atc aca aca actctt gaa ttt gcg atg cag gat 816 Glu Lys Leu Gly Phe Ile Thr Thr Thr LeuGlu Phe Ala Met Gln Asp 260 265 270 aaa gag ctt cgc gat cag ctc gtt ccattt atg gaa ggt tta cta aac 864 Lys Glu Leu Arg Asp Gln Leu Val Pro PheMet Glu Gly Leu Leu Asn 275 280 285 aaa gaa gaa atc 876 Lys Glu Glu Ile290 22 292 PRT Bacillus subtilis 22 Met Lys Lys Val Arg Lys Ala Ile IlePro Ala Ala Gly Leu Gly Thr 1 5 10 15 Arg Phe Leu Pro Ala Thr Lys AlaMet Pro Lys Glu Met Leu Pro Ile 20 25 30 Val Asp Lys Pro Thr Ile Gln TyrIle Ile Glu Glu Ala Val Glu Ala 35 40 45 Gly Ile Glu Asp Ile Ile Ile ValThr Gly Lys Ser Lys Arg Ala Ile 50 55 60 Glu Asp His Phe Asp Tyr Ser ProGlu Leu Glu Arg Asn Leu Glu Glu 65 70 75 80 Lys Gly Lys Thr Glu Leu LeuGlu Lys Val Lys Lys Ala Ser Asn Leu 85 90 95 Ala Asp Ile His Tyr Ile ArgGln Lys Glu Pro Lys Gly Leu Gly His 100 105 110 Ala Val Trp Cys Ala ArgAsn Phe Ile Gly Asp Glu Pro Phe Ala Val 115 120 125 Leu Leu Gly Asp AspIle Val Gln Ala Glu Thr Pro Gly Leu Arg Gln 130 135 140 Leu Met Asp GluTyr Glu Lys Thr Leu Ser Ser Ile Ile Gly Val Gln 145 150 155 160 Gln ValPro Glu Glu Glu Thr His Arg Tyr Gly Ile Ile Asp Pro Leu 165 170 175 ThrSer Glu Gly Arg Arg Tyr Gln Val Lys Asn Phe Val Glu Lys Pro 180 185 190Pro Lys Gly Thr Ala Pro Ser Asn Leu Ala Ile Leu Gly Arg Tyr Val 195 200205 Phe Thr Pro Glu Ile Phe Met Tyr Leu Glu Glu Gln Gln Val Gly Ala 210215 220 Gly Gly Glu Ile Gln Leu Thr Asp Ala Ile Gln Lys Leu Asn Glu Ile225 230 235 240 Gln Arg Val Phe Ala Tyr Asp Phe Glu Gly Lys Arg Tyr AspVal Gly 245 250 255 Glu Lys Leu Gly Phe Ile Thr Thr Thr Leu Glu Phe AlaMet Gln Asp 260 265 270 Lys Glu Leu Arg Asp Gln Leu Val Pro Phe Met GluGly Leu Leu Asn 275 280 285 Lys Glu Glu Ile 290 23 27 DNA Bacillussubtilis 23 tctagatttt tcgatcataa ggaaggt 27 24 49 DNA Bacillus subtilis24 gttaacgaat tccagctatg taggatccaa tgtccaatag cctttttgt 49 25 20 DNABacillus subtilis 25 aaaaaggctt ctaacctggc 20 26 20 DNA Bacillussubtilis 26 aaaccgccta aaggcacagc 20 27 20 DNA Bacillus subtilis 27gccaggttag aagccttttt 20 28 20 DNA Bacillus subtilis 28 gctgtgcctttaggcggttt 20 29 1368 DNA Bacillus subtilis CDS (1)..(1368) 29 atg gataag cgg ttt gca gtt gtt tta gcg gct gga caa gga acg aga 48 Met Asp LysArg Phe Ala Val Val Leu Ala Ala Gly Gln Gly Thr Arg 1 5 10 15 atg aaatcg aag ctt tat aaa gtc ctt cat cca gtt tgc ggt aag cct 96 Met Lys SerLys Leu Tyr Lys Val Leu His Pro Val Cys Gly Lys Pro 20 25 30 atg gta gagcac gtc gtg gac gaa gcc tta aaa tta tct tta tca aag 144 Met Val Glu HisVal Val Asp Glu Ala Leu Lys Leu Ser Leu Ser Lys 35 40 45 ctt gtc acg attgtc gga cat ggt gcg gaa gaa gtg aaa aag cag ctt 192 Leu Val Thr Ile ValGly His Gly Ala Glu Glu Val Lys Lys Gln Leu 50 55 60 ggt gat aaa agc gagtac gcg ctt caa gca aaa cag ctt ggc act gct 240 Gly Asp Lys Ser Glu TyrAla Leu Gln Ala Lys Gln Leu Gly Thr Ala 65 70 75 80 cat gct gta aaa caggca cag cca ttt ctt gct gac gaa aaa ggc gtc 288 His Ala Val Lys Gln AlaGln Pro Phe Leu Ala Asp Glu Lys Gly Val 85 90 95 aca att gtc att tgc ggagat acg ccg ctt ttg aca gca gag acg atg 336 Thr Ile Val Ile Cys Gly AspThr Pro Leu Leu Thr Ala Glu Thr Met 100 105 110 gaa cag atg ctg aaa gaacat aca caa aga gaa gcg aaa gct acg att 384 Glu Gln Met Leu Lys Glu HisThr Gln Arg Glu Ala Lys Ala Thr Ile 115 120 125 tta act gcg gtt gca gaagat cca act gga tac ggc cgc att att cgc 432 Leu Thr Ala Val Ala Glu AspPro Thr Gly Tyr Gly Arg Ile Ile Arg 130 135 140 agc gaa aac gga gcg gttcaa aaa ata gtt gag cat aag gac gcc tct 480 Ser Glu Asn Gly Ala Val GlnLys Ile Val Glu His Lys Asp Ala Ser 145 150 155 160 gaa gaa gaa cgt cttgta act gag atc aac acc ggt acg tat tgt ttt 528 Glu Glu Glu Arg Leu ValThr Glu Ile Asn Thr Gly Thr Tyr Cys Phe 165 170 175 gac aat gaa gcg ctattt cgg gct att gat cag gtg tct aat gat aat 576 Asp Asn Glu Ala Leu PheArg Ala Ile Asp Gln Val Ser Asn Asp Asn 180 185 190 gca caa ggc gag tattat ttg ccg gat gtc ata gag att ctt aaa aat 624 Ala Gln Gly Glu Tyr TyrLeu Pro Asp Val Ile Glu Ile Leu Lys Asn 195 200 205 gaa ggc gaa act gttgcc gct tac cag act ggt aat ttc caa gaa acg 672 Glu Gly Glu Thr Val AlaAla Tyr Gln Thr Gly Asn Phe Gln Glu Thr 210 215 220 ctc gga gtt aat gataga gtt gct ctt tct cag gca gaa caa ttt atg 720 Leu Gly Val Asn Asp ArgVal Ala Leu Ser Gln Ala Glu Gln Phe Met 225 230 235 240 aaa gag cgc attaat aaa cgg cat atg caa aat ggc gtg acg ttg att 768 Lys Glu Arg Ile AsnLys Arg His Met Gln Asn Gly Val Thr Leu Ile 245 250 255 gac ccg atg aatacg tat att tct cct gac gct gtt atc gga agc gat 816 Asp Pro Met Asn ThrTyr Ile Ser Pro Asp Ala Val Ile Gly Ser Asp 260 265 270 act gtg att taccct gga act gtg att aaa ggt gag gtg caa atc gga 864 Thr Val Ile Tyr ProGly Thr Val Ile Lys Gly Glu Val Gln Ile Gly 275 280 285 gaa gat acg attatt ggc cct cat acg gag att atg aat agt gcc att 912 Glu Asp Thr Ile IleGly Pro His Thr Glu Ile Met Asn Ser Ala Ile 290 295 300 ggc agc cgt acggtt att aaa caa tcg gta gtc aat cac agt aaa gtg 960 Gly Ser Arg Thr ValIle Lys Gln Ser Val Val Asn His Ser Lys Val 305 310 315 320 ggg aat gatgta aac ata gga cct ttt gct cac atc aga cct gat tct 1008 Gly Asn Asp ValAsn Ile Gly Pro Phe Ala His Ile Arg Pro Asp Ser 325 330 335 gtc atc gggaat gaa gtg aag atc ggg aat ttt gta gaa att aaa aag 1056 Val Ile Gly AsnGlu Val Lys Ile Gly Asn Phe Val Glu Ile Lys Lys 340 345 350 act caa ttcgga gac cga agc aag gca tct cat cta agc tat gtc ggc 1104 Thr Gln Phe GlyAsp Arg Ser Lys Ala Ser His Leu Ser Tyr Val Gly 355 360 365 gat gct gaggta ggc act gat gta aac ctg ggc tgc ggt tca att act 1152 Asp Ala Glu ValGly Thr Asp Val Asn Leu Gly Cys Gly Ser Ile Thr 370 375 380 gtc aat tatgat gga aag aat aag tat ttg aca aaa att gaa gat ggc 1200 Val Asn Tyr AspGly Lys Asn Lys Tyr Leu Thr Lys Ile Glu Asp Gly 385 390 395 400 gcg tttatc ggc tgc aat tcc aac ttg gtt gcc cct gtc aca gtc gga 1248 Ala Phe IleGly Cys Asn Ser Asn Leu Val Ala Pro Val Thr Val Gly 405 410 415 gaa ggcgct tat gtg gcg gca ggt tca act gtt acg gaa gat gta cct 1296 Glu Gly AlaTyr Val Ala Ala Gly Ser Thr Val Thr Glu Asp Val Pro 420 425 430 gga aaagca ctt gct att gcc aga gcg aga caa gta aat aaa gac gat 1344 Gly Lys AlaLeu Ala Ile Ala Arg Ala Arg Gln Val Asn Lys Asp Asp 435 440 445 tat gtgaaa aat att cat aaa aaa 1368 Tyr Val Lys Asn Ile His Lys Lys 450 455 30456 PRT Bacillus subtilis 30 Met Asp Lys Arg Phe Ala Val Val Leu Ala AlaGly Gln Gly Thr Arg 1 5 10 15 Met Lys Ser Lys Leu Tyr Lys Val Leu HisPro Val Cys Gly Lys Pro 20 25 30 Met Val Glu His Val Val Asp Glu Ala LeuLys Leu Ser Leu Ser Lys 35 40 45 Leu Val Thr Ile Val Gly His Gly Ala GluGlu Val Lys Lys Gln Leu 50 55 60 Gly Asp Lys Ser Glu Tyr Ala Leu Gln AlaLys Gln Leu Gly Thr Ala 65 70 75 80 His Ala Val Lys Gln Ala Gln Pro PheLeu Ala Asp Glu Lys Gly Val 85 90 95 Thr Ile Val Ile Cys Gly Asp Thr ProLeu Leu Thr Ala Glu Thr Met 100 105 110 Glu Gln Met Leu Lys Glu His ThrGln Arg Glu Ala Lys Ala Thr Ile 115 120 125 Leu Thr Ala Val Ala Glu AspPro Thr Gly Tyr Gly Arg Ile Ile Arg 130 135 140 Ser Glu Asn Gly Ala ValGln Lys Ile Val Glu His Lys Asp Ala Ser 145 150 155 160 Glu Glu Glu ArgLeu Val Thr Glu Ile Asn Thr Gly Thr Tyr Cys Phe 165 170 175 Asp Asn GluAla Leu Phe Arg Ala Ile Asp Gln Val Ser Asn Asp Asn 180 185 190 Ala GlnGly Glu Tyr Tyr Leu Pro Asp Val Ile Glu Ile Leu Lys Asn 195 200 205 GluGly Glu Thr Val Ala Ala Tyr Gln Thr Gly Asn Phe Gln Glu Thr 210 215 220Leu Gly Val Asn Asp Arg Val Ala Leu Ser Gln Ala Glu Gln Phe Met 225 230235 240 Lys Glu Arg Ile Asn Lys Arg His Met Gln Asn Gly Val Thr Leu Ile245 250 255 Asp Pro Met Asn Thr Tyr Ile Ser Pro Asp Ala Val Ile Gly SerAsp 260 265 270 Thr Val Ile Tyr Pro Gly Thr Val Ile Lys Gly Glu Val GlnIle Gly 275 280 285 Glu Asp Thr Ile Ile Gly Pro His Thr Glu Ile Met AsnSer Ala Ile 290 295 300 Gly Ser Arg Thr Val Ile Lys Gln Ser Val Val AsnHis Ser Lys Val 305 310 315 320 Gly Asn Asp Val Asn Ile Gly Pro Phe AlaHis Ile Arg Pro Asp Ser 325 330 335 Val Ile Gly Asn Glu Val Lys Ile GlyAsn Phe Val Glu Ile Lys Lys 340 345 350 Thr Gln Phe Gly Asp Arg Ser LysAla Ser His Leu Ser Tyr Val Gly 355 360 365 Asp Ala Glu Val Gly Thr AspVal Asn Leu Gly Cys Gly Ser Ile Thr 370 375 380 Val Asn Tyr Asp Gly LysAsn Lys Tyr Leu Thr Lys Ile Glu Asp Gly 385 390 395 400 Ala Phe Ile GlyCys Asn Ser Asn Leu Val Ala Pro Val Thr Val Gly 405 410 415 Glu Gly AlaTyr Val Ala Ala Gly Ser Thr Val Thr Glu Asp Val Pro 420 425 430 Gly LysAla Leu Ala Ile Ala Arg Ala Arg Gln Val Asn Lys Asp Asp 435 440 445 TyrVal Lys Asn Ile His Lys Lys 450 455 31 26 DNA Bacillus subtilis 31ggatcctttc tatggataaa agggat 26 32 31 DNA Bacillus subtilis 32gttaacagga ttatttttta tgaatatttt t 31 33 20 DNA Bacillus subtilis 33cagagacgat ggaacagatg 20 34 20 DNA Bacillus subtilis 34 ggagttaatgatagagttgc 20 35 20 DNA Bacillus subtilis 35 gaagatcggg aattttgtag 20 3620 DNA Bacillus subtilis 36 catctgttcc atcgtctctg 20 37 20 DNA Bacillussubtilis 37 gcaactctat cattaactcc 20 38 20 DNA Bacillus subtilis 38ctacaaaatt cccgatcttc 20 39 54 DNA Streptococcus equisimilis 39gtgtcggaac attcattaca tgcttaagca cccgctgtcc ttcttgttat ctcc 54 40 1203DNA Streptococcus equisimilis CDS (1)..(1203) 40 gtg aaa att tct gta gcaggc tca gga tat gtc ggc cta tcc ttg agt 48 Val Lys Ile Ser Val Ala GlySer Gly Tyr Val Gly Leu Ser Leu Ser 1 5 10 15 att tta ctg gca caa cataat gac gtc act gtt gtt gat att att gat 96 Ile Leu Leu Ala Gln His AsnAsp Val Thr Val Val Asp Ile Ile Asp 20 25 30 gaa aag gtg aga ttg atc aatcaa ggc ata tct cca atc aag gat gct 144 Glu Lys Val Arg Leu Ile Asn GlnGly Ile Ser Pro Ile Lys Asp Ala 35 40 45 gat att gag gag tat tta aaa aatgcg ccg cta aat ctc aca gcg acc 192 Asp Ile Glu Glu Tyr Leu Lys Asn AlaPro Leu Asn Leu Thr Ala Thr 50 55 60 ctt gat ggc gca agc gct tat agc aatgca gac ctt att atc att gct 240 Leu Asp Gly Ala Ser Ala Tyr Ser Asn AlaAsp Leu Ile Ile Ile Ala 65 70 75 80 act ccg aca aat tat gac agc gaa cgcaac tac ttt gac aca agg cat 288 Thr Pro Thr Asn Tyr Asp Ser Glu Arg AsnTyr Phe Asp Thr Arg His 85 90 95 gtt gaa gag gtc att gag cag gtc cta gaccta aat gcg tca gca acc 336 Val Glu Glu Val Ile Glu Gln Val Leu Asp LeuAsn Ala Ser Ala Thr 100 105 110 att att atc aaa tca acc ata cca cta ggcttt atc aag cat gtt agg 384 Ile Ile Ile Lys Ser Thr Ile Pro Leu Gly PheIle Lys His Val Arg 115 120 125 gaa aaa tac cag aca gat cgt att att tttagc cca gaa ttt tta aga 432 Glu Lys Tyr Gln Thr Asp Arg Ile Ile Phe SerPro Glu Phe Leu Arg 130 135 140 gaa tca aaa gcc tta tac gat aac ctt taccca agt cgg atc att gtt 480 Glu Ser Lys Ala Leu Tyr Asp Asn Leu Tyr ProSer Arg Ile Ile Val 145 150 155 160 tct tat gaa aag gac gac tca cca agggtt att cag gct gct aaa gcc 528 Ser Tyr Glu Lys Asp Asp Ser Pro Arg ValIle Gln Ala Ala Lys Ala 165 170 175 ttt gct ggt ctt tta aag gaa gga gccaaa agc aag gat act ccg gtc 576 Phe Ala Gly Leu Leu Lys Glu Gly Ala LysSer Lys Asp Thr Pro Val 180 185 190 tta ttt atg ggc tca cag gag gct gaggcg gtc aag cta ttt gcg aat 624 Leu Phe Met Gly Ser Gln Glu Ala Glu AlaVal Lys Leu Phe Ala Asn 195 200 205 acc ttt ttg gct atg cgg gtg tct tacttt aat gaa tta gac acc tat 672 Thr Phe Leu Ala Met Arg Val Ser Tyr PheAsn Glu Leu Asp Thr Tyr 210 215 220 tcc gaa agc aag ggt cta gat gct cagcgc gtg att gaa gga gtc tgt 720 Ser Glu Ser Lys Gly Leu Asp Ala Gln ArgVal Ile Glu Gly Val Cys 225 230 235 240 cat gat cag cgc att ggt aac cattac aat aac cct tcc ttt gga tat 768 His Asp Gln Arg Ile Gly Asn His TyrAsn Asn Pro Ser Phe Gly Tyr 245 250 255 ggc ggc tat tgc ctg cca aag gacagc aaa cag ctg ttg gca aat tat 816 Gly Gly Tyr Cys Leu Pro Lys Asp SerLys Gln Leu Leu Ala Asn Tyr 260 265 270 aga ggc att ccc cag tcc ttg atgtca gcg att gtt gag tcc aac aag 864 Arg Gly Ile Pro Gln Ser Leu Met SerAla Ile Val Glu Ser Asn Lys 275 280 285 ata cga aaa tcc tat tta gct gaacaa ata tta gac aga gcc tct agt 912 Ile Arg Lys Ser Tyr Leu Ala Glu GlnIle Leu Asp Arg Ala Ser Ser 290 295 300 caa aag cag gct ggt gta cca ttaacg att ggc ttt tac cgc ttg att 960 Gln Lys Gln Ala Gly Val Pro Leu ThrIle Gly Phe Tyr Arg Leu Ile 305 310 315 320 atg aaa agc aac tct gat aatttc cga gaa agc gcc att aaa gat att 1008 Met Lys Ser Asn Ser Asp Asn PheArg Glu Ser Ala Ile Lys Asp Ile 325 330 335 att gat atc atc aac gac tatggg gtt aat att gtc att tac gaa ccc 1056 Ile Asp Ile Ile Asn Asp Tyr GlyVal Asn Ile Val Ile Tyr Glu Pro 340 345 350 atg ctt ggc gag gat att ggctac agg gtt gtc aag gac tta gag cag 1104 Met Leu Gly Glu Asp Ile Gly TyrArg Val Val Lys Asp Leu Glu Gln 355 360 365 ttc aaa aac gag tct aca atcatt gtg tca aat cgc ttt gag gac gac 1152 Phe Lys Asn Glu Ser Thr Ile IleVal Ser Asn Arg Phe Glu Asp Asp 370 375 380 cta gga gat gtc att gat aaggtt tat acg aga gat gtc ttt gga aga 1200 Leu Gly Asp Val Ile Asp Lys ValTyr Thr Arg Asp Val Phe Gly Arg 385 390 395 400 gac 1203 Asp 41 401 PRTStreptococcus equisimilis 41 Val Lys Ile Ser Val Ala Gly Ser Gly Tyr ValGly Leu Ser Leu Ser 1 5 10 15 Ile Leu Leu Ala Gln His Asn Asp Val ThrVal Val Asp Ile Ile Asp 20 25 30 Glu Lys Val Arg Leu Ile Asn Gln Gly IleSer Pro Ile Lys Asp Ala 35 40 45 Asp Ile Glu Glu Tyr Leu Lys Asn Ala ProLeu Asn Leu Thr Ala Thr 50 55 60 Leu Asp Gly Ala Ser Ala Tyr Ser Asn AlaAsp Leu Ile Ile Ile Ala 65 70 75 80 Thr Pro Thr Asn Tyr Asp Ser Glu ArgAsn Tyr Phe Asp Thr Arg His 85 90 95 Val Glu Glu Val Ile Glu Gln Val LeuAsp Leu Asn Ala Ser Ala Thr 100 105 110 Ile Ile Ile Lys Ser Thr Ile ProLeu Gly Phe Ile Lys His Val Arg 115 120 125 Glu Lys Tyr Gln Thr Asp ArgIle Ile Phe Ser Pro Glu Phe Leu Arg 130 135 140 Glu Ser Lys Ala Leu TyrAsp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145 150 155 160 Ser Tyr Glu LysAsp Asp Ser Pro Arg Val Ile Gln Ala Ala Lys Ala 165 170 175 Phe Ala GlyLeu Leu Lys Glu Gly Ala Lys Ser Lys Asp Thr Pro Val 180 185 190 Leu PheMet Gly Ser Gln Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195 200 205 ThrPhe Leu Ala Met Arg Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210 215 220Ser Glu Ser Lys Gly Leu Asp Ala Gln Arg Val Ile Glu Gly Val Cys 225 230235 240 His Asp Gln Arg Ile Gly Asn His Tyr Asn Asn Pro Ser Phe Gly Tyr245 250 255 Gly Gly Tyr Cys Leu Pro Lys Asp Ser Lys Gln Leu Leu Ala AsnTyr 260 265 270 Arg Gly Ile Pro Gln Ser Leu Met Ser Ala Ile Val Glu SerAsn Lys 275 280 285 Ile Arg Lys Ser Tyr Leu Ala Glu Gln Ile Leu Asp ArgAla Ser Ser 290 295 300 Gln Lys Gln Ala Gly Val Pro Leu Thr Ile Gly PheTyr Arg Leu Ile 305 310 315 320 Met Lys Ser Asn Ser Asp Asn Phe Arg GluSer Ala Ile Lys Asp Ile 325 330 335 Ile Asp Ile Ile Asn Asp Tyr Gly ValAsn Ile Val Ile Tyr Glu Pro 340 345 350 Met Leu Gly Glu Asp Ile Gly TyrArg Val Val Lys Asp Leu Glu Gln 355 360 365 Phe Lys Asn Glu Ser Thr IleIle Val Ser Asn Arg Phe Glu Asp Asp 370 375 380 Leu Gly Asp Val Ile AspLys Val Tyr Thr Arg Asp Val Phe Gly Arg 385 390 395 400 Asp 42 900 DNAStreptococcus equisimilis CDS (1)..(900) 42 atg aca aag gtc aga aaa gccatt atc cca gcc gcc ggc cta ggc act 48 Met Thr Lys Val Arg Lys Ala IleIle Pro Ala Ala Gly Leu Gly Thr 1 5 10 15 cgc ttc cta ccc gcc acc aaggca ctg gcc aag gaa atg ctc cca atc 96 Arg Phe Leu Pro Ala Thr Lys AlaLeu Ala Lys Glu Met Leu Pro Ile 20 25 30 gtc gat aag cca acc att caa ttcatc gtc gag gaa gct cta aag gcc 144 Val Asp Lys Pro Thr Ile Gln Phe IleVal Glu Glu Ala Leu Lys Ala 35 40 45 ggt atc gag gag att ctt gtc gtc accggc aag gcc aaa cgc tct att 192 Gly Ile Glu Glu Ile Leu Val Val Thr GlyLys Ala Lys Arg Ser Ile 50 55 60 gaa gac cac ttt gac tcc aac ttc gag ctcgaa tac aat ctc caa gcc 240 Glu Asp His Phe Asp Ser Asn Phe Glu Leu GluTyr Asn Leu Gln Ala 65 70 75 80 aag ggc aaa acc gag ctg ctc aag ctc gttgat gag acc act gcc atc 288 Lys Gly Lys Thr Glu Leu Leu Lys Leu Val AspGlu Thr Thr Ala Ile 85 90 95 aac ctg cac ttc att cgt cag agc cac cct agagga cta ggg gac gct 336 Asn Leu His Phe Ile Arg Gln Ser His Pro Arg GlyLeu Gly Asp Ala 100 105 110 gtc ctc cag gcc aag gcc ttt gtg ggc aat gagccc ttt gtg gtc atg 384 Val Leu Gln Ala Lys Ala Phe Val Gly Asn Glu ProPhe Val Val Met 115 120 125 ctg ggg gat gac ctc atg gat att acc aat cctagt gcc aag ccc ttg 432 Leu Gly Asp Asp Leu Met Asp Ile Thr Asn Pro SerAla Lys Pro Leu 130 135 140 gcc aag cag ctc att gag gat tat gat tgc acacac gcc tca acg att 480 Ala Lys Gln Leu Ile Glu Asp Tyr Asp Cys Thr HisAla Ser Thr Ile 145 150 155 160 gca gtg atg agg gtg ccg cat gag gag gtttcc aat tat ggc gtg att 528 Ala Val Met Arg Val Pro His Glu Glu Val SerAsn Tyr Gly Val Ile 165 170 175 gca ccg caa ggg aag gct gtt aag ggc ttgtat agt gtg gag acc ttt 576 Ala Pro Gln Gly Lys Ala Val Lys Gly Leu TyrSer Val Glu Thr Phe 180 185 190 gtt gag aag cca agt cca gat gag gca ccgagt gac tta gcg att att 624 Val Glu Lys Pro Ser Pro Asp Glu Ala Pro SerAsp Leu Ala Ile Ile 195 200 205 ggt cga tat ttg ttg acg cct gag att tttgcc ata ttg gag aat cag 672 Gly Arg Tyr Leu Leu Thr Pro Glu Ile Phe AlaIle Leu Glu Asn Gln 210 215 220 gcg cct ggg gct ggc aat gag gta cag ctagcc gat gcg att gac aag 720 Ala Pro Gly Ala Gly Asn Glu Val Gln Leu AlaAsp Ala Ile Asp Lys 225 230 235 240 ctc aac aag act cag cgg gtt ttt gcgagg gag ttt aag gga gag cgg 768 Leu Asn Lys Thr Gln Arg Val Phe Ala ArgGlu Phe Lys Gly Glu Arg 245 250 255 tat gat gtt ggg gac aag ttt ggc tttatg aag acc tca ctt gac tat 816 Tyr Asp Val Gly Asp Lys Phe Gly Phe MetLys Thr Ser Leu Asp Tyr 260 265 270 gct ctc aag cac cct cag gtc aag gacgac ctc act gac tac att ata 864 Ala Leu Lys His Pro Gln Val Lys Asp AspLeu Thr Asp Tyr Ile Ile 275 280 285 aag ctc agt aag caa ctg aac aag gacgtt aaa aaa 900 Lys Leu Ser Lys Gln Leu Asn Lys Asp Val Lys Lys 290 295300 43 300 PRT Streptococcus equisimilis 43 Met Thr Lys Val Arg Lys AlaIle Ile Pro Ala Ala Gly Leu Gly Thr 1 5 10 15 Arg Phe Leu Pro Ala ThrLys Ala Leu Ala Lys Glu Met Leu Pro Ile 20 25 30 Val Asp Lys Pro Thr IleGln Phe Ile Val Glu Glu Ala Leu Lys Ala 35 40 45 Gly Ile Glu Glu Ile LeuVal Val Thr Gly Lys Ala Lys Arg Ser Ile 50 55 60 Glu Asp His Phe Asp SerAsn Phe Glu Leu Glu Tyr Asn Leu Gln Ala 65 70 75 80 Lys Gly Lys Thr GluLeu Leu Lys Leu Val Asp Glu Thr Thr Ala Ile 85 90 95 Asn Leu His Phe IleArg Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100 105 110 Val Leu Gln AlaLys Ala Phe Val Gly Asn Glu Pro Phe Val Val Met 115 120 125 Leu Gly AspAsp Leu Met Asp Ile Thr Asn Pro Ser Ala Lys Pro Leu 130 135 140 Ala LysGln Leu Ile Glu Asp Tyr Asp Cys Thr His Ala Ser Thr Ile 145 150 155 160Ala Val Met Arg Val Pro His Glu Glu Val Ser Asn Tyr Gly Val Ile 165 170175 Ala Pro Gln Gly Lys Ala Val Lys Gly Leu Tyr Ser Val Glu Thr Phe 180185 190 Val Glu Lys Pro Ser Pro Asp Glu Ala Pro Ser Asp Leu Ala Ile Ile195 200 205 Gly Arg Tyr Leu Leu Thr Pro Glu Ile Phe Ala Ile Leu Glu AsnGln 210 215 220 Ala Pro Gly Ala Gly Asn Glu Val Gln Leu Ala Asp Ala IleAsp Lys 225 230 235 240 Leu Asn Lys Thr Gln Arg Val Phe Ala Arg Glu PheLys Gly Glu Arg 245 250 255 Tyr Asp Val Gly Asp Lys Phe Gly Phe Met LysThr Ser Leu Asp Tyr 260 265 270 Ala Leu Lys His Pro Gln Val Lys Asp AspLeu Thr Asp Tyr Ile Ile 275 280 285 Lys Leu Ser Lys Gln Leu Asn Lys AspVal Lys Lys 290 295 300 44 1380 DNA Streptococcus equisimilis CDS(1)..(1380) 44 atg aaa aac tac gcc att atc cta gca gct gga aag gga acccgc atg 48 Met Lys Asn Tyr Ala Ile Ile Leu Ala Ala Gly Lys Gly Thr ArgMet 1 5 10 15 aat tca ggg ctt tcc aag gtg ctg cac aag gta tca ggc ctaagc atg 96 Asn Ser Gly Leu Ser Lys Val Leu His Lys Val Ser Gly Leu SerMet 20 25 30 ctg gag cat gtc ctc aag agc gtc tca gcc cta gct cct caa aagcaa 144 Leu Glu His Val Leu Lys Ser Val Ser Ala Leu Ala Pro Gln Lys Gln35 40 45 ctc aca gtg atc ggt cat cag gca gag caa gta cgt gcc gtc cta ggt192 Leu Thr Val Ile Gly His Gln Ala Glu Gln Val Arg Ala Val Leu Gly 5055 60 gat caa tta ctg aca gtg gtg caa gag gag cag cta gga aca ggc cat240 Asp Gln Leu Leu Thr Val Val Gln Glu Glu Gln Leu Gly Thr Gly His 6570 75 80 gca gtc atg atg gca gaa gag gag cta tct ggc tta gaa ggg cag acc288 Ala Val Met Met Ala Glu Glu Glu Leu Ser Gly Leu Glu Gly Gln Thr 8590 95 cta gtg att gca ggt gac acc ccc ttg atc aga gga gaa agc ctc aag336 Leu Val Ile Ala Gly Asp Thr Pro Leu Ile Arg Gly Glu Ser Leu Lys 100105 110 gct ctg cta gac tat cat atc aga gaa aag aat gtg gca acc att ctc384 Ala Leu Leu Asp Tyr His Ile Arg Glu Lys Asn Val Ala Thr Ile Leu 115120 125 aca gcc aat gcc aag gat ccc ttt ggc tac ggc cga atc att cgc aat432 Thr Ala Asn Ala Lys Asp Pro Phe Gly Tyr Gly Arg Ile Ile Arg Asn 130135 140 gca gca gga gag gtg gtc aac atc gtt gaa caa aag gac gct aat gag480 Ala Ala Gly Glu Val Val Asn Ile Val Glu Gln Lys Asp Ala Asn Glu 145150 155 160 gca gag caa gag gtc aag gag atc aac aca ggg acc tat atc tttgac 528 Ala Glu Gln Glu Val Lys Glu Ile Asn Thr Gly Thr Tyr Ile Phe Asp165 170 175 aat aag cgc ctc ttt gag gct cta aag cat ctc acg act gat aatgcc 576 Asn Lys Arg Leu Phe Glu Ala Leu Lys His Leu Thr Thr Asp Asn Ala180 185 190 caa ggg gaa tat tac cta acc gat gtg atc agt att ttc aag gccagc 624 Gln Gly Glu Tyr Tyr Leu Thr Asp Val Ile Ser Ile Phe Lys Ala Ser195 200 205 caa gaa aag gtt gga gct tac ctg ctg aag gat ttt gat gaa agccta 672 Gln Glu Lys Val Gly Ala Tyr Leu Leu Lys Asp Phe Asp Glu Ser Leu210 215 220 ggg gtt aat gat cgc cta gct cta gcc cag gct gag gtg atc atgcag 720 Gly Val Asn Asp Arg Leu Ala Leu Ala Gln Ala Glu Val Ile Met Gln225 230 235 240 gag cgg atc aac aag cag cac atg ctt aat ggg gtg acc ctgcaa aac 768 Glu Arg Ile Asn Lys Gln His Met Leu Asn Gly Val Thr Leu GlnAsn 245 250 255 cct gca gct acc tat atc gaa agc agt gta gag att gcg ccggac gtc 816 Pro Ala Ala Thr Tyr Ile Glu Ser Ser Val Glu Ile Ala Pro AspVal 260 265 270 ttg att gaa gct aat gtg acc cta aag gga cag act aga attggc agc 864 Leu Ile Glu Ala Asn Val Thr Leu Lys Gly Gln Thr Arg Ile GlySer 275 280 285 aga agt gtt ata acc aat ggg agc tat atc ctt gat tca aggctt ggt 912 Arg Ser Val Ile Thr Asn Gly Ser Tyr Ile Leu Asp Ser Arg LeuGly 290 295 300 gag ggc gta gtg gtg agc cag tca gtg att gag ggc tca gtccta gca 960 Glu Gly Val Val Val Ser Gln Ser Val Ile Glu Gly Ser Val LeuAla 305 310 315 320 gat ggt gtg aca gta ggg ccc tat gca cac att cgc ccggac tct cag 1008 Asp Gly Val Thr Val Gly Pro Tyr Ala His Ile Arg Pro AspSer Gln 325 330 335 ctc gat gag tgt gtt cat att ggg aac ttt gta gag gttaag ggg tct 1056 Leu Asp Glu Cys Val His Ile Gly Asn Phe Val Glu Val LysGly Ser 340 345 350 cat cta ggg gcc aat acc aag gca ggg cat ttg act tatctg ggg aat 1104 His Leu Gly Ala Asn Thr Lys Ala Gly His Leu Thr Tyr LeuGly Asn 355 360 365 gcc gag att ggc tca gag gtt aat att ggt gca gga agcatt acg gtt 1152 Ala Glu Ile Gly Ser Glu Val Asn Ile Gly Ala Gly Ser IleThr Val 370 375 380 aat tat gat ggt caa cgg aaa tac cag aca gtg att ggcgat cac gct 1200 Asn Tyr Asp Gly Gln Arg Lys Tyr Gln Thr Val Ile Gly AspHis Ala 385 390 395 400 ttt att ggg agt cat tcg act ttg ata gct ccg gtagag gtt ggg gag 1248 Phe Ile Gly Ser His Ser Thr Leu Ile Ala Pro Val GluVal Gly Glu 405 410 415 aat gct tta aca gca gca ggg tct acg ata gcc cagtcg gtg cca gca 1296 Asn Ala Leu Thr Ala Ala Gly Ser Thr Ile Ala Gln SerVal Pro Ala 420 425 430 gac agt gtg gct ata ggg cgt agc cgt cag gtg gtgaag gaa ggc tat 1344 Asp Ser Val Ala Ile Gly Arg Ser Arg Gln Val Val LysGlu Gly Tyr 435 440 445 gcc aag agg cta cca cat cac ccg gat cag ccc cag1380 Ala Lys Arg Leu Pro His His Pro Asp Gln Pro Gln 450 455 460 45 460PRT Streptococcus equisimilis 45 Met Lys Asn Tyr Ala Ile Ile Leu Ala AlaGly Lys Gly Thr Arg Met 1 5 10 15 Asn Ser Gly Leu Ser Lys Val Leu HisLys Val Ser Gly Leu Ser Met 20 25 30 Leu Glu His Val Leu Lys Ser Val SerAla Leu Ala Pro Gln Lys Gln 35 40 45 Leu Thr Val Ile Gly His Gln Ala GluGln Val Arg Ala Val Leu Gly 50 55 60 Asp Gln Leu Leu Thr Val Val Gln GluGlu Gln Leu Gly Thr Gly His 65 70 75 80 Ala Val Met Met Ala Glu Glu GluLeu Ser Gly Leu Glu Gly Gln Thr 85 90 95 Leu Val Ile Ala Gly Asp Thr ProLeu Ile Arg Gly Glu Ser Leu Lys 100 105 110 Ala Leu Leu Asp Tyr His IleArg Glu Lys Asn Val Ala Thr Ile Leu 115 120 125 Thr Ala Asn Ala Lys AspPro Phe Gly Tyr Gly Arg Ile Ile Arg Asn 130 135 140 Ala Ala Gly Glu ValVal Asn Ile Val Glu Gln Lys Asp Ala Asn Glu 145 150 155 160 Ala Glu GlnGlu Val Lys Glu Ile Asn Thr Gly Thr Tyr Ile Phe Asp 165 170 175 Asn LysArg Leu Phe Glu Ala Leu Lys His Leu Thr Thr Asp Asn Ala 180 185 190 GlnGly Glu Tyr Tyr Leu Thr Asp Val Ile Ser Ile Phe Lys Ala Ser 195 200 205Gln Glu Lys Val Gly Ala Tyr Leu Leu Lys Asp Phe Asp Glu Ser Leu 210 215220 Gly Val Asn Asp Arg Leu Ala Leu Ala Gln Ala Glu Val Ile Met Gln 225230 235 240 Glu Arg Ile Asn Lys Gln His Met Leu Asn Gly Val Thr Leu GlnAsn 245 250 255 Pro Ala Ala Thr Tyr Ile Glu Ser Ser Val Glu Ile Ala ProAsp Val 260 265 270 Leu Ile Glu Ala Asn Val Thr Leu Lys Gly Gln Thr ArgIle Gly Ser 275 280 285 Arg Ser Val Ile Thr Asn Gly Ser Tyr Ile Leu AspSer Arg Leu Gly 290 295 300 Glu Gly Val Val Val Ser Gln Ser Val Ile GluGly Ser Val Leu Ala 305 310 315 320 Asp Gly Val Thr Val Gly Pro Tyr AlaHis Ile Arg Pro Asp Ser Gln 325 330 335 Leu Asp Glu Cys Val His Ile GlyAsn Phe Val Glu Val Lys Gly Ser 340 345 350 His Leu Gly Ala Asn Thr LysAla Gly His Leu Thr Tyr Leu Gly Asn 355 360 365 Ala Glu Ile Gly Ser GluVal Asn Ile Gly Ala Gly Ser Ile Thr Val 370 375 380 Asn Tyr Asp Gly GlnArg Lys Tyr Gln Thr Val Ile Gly Asp His Ala 385 390 395 400 Phe Ile GlySer His Ser Thr Leu Ile Ala Pro Val Glu Val Gly Glu 405 410 415 Asn AlaLeu Thr Ala Ala Gly Ser Thr Ile Ala Gln Ser Val Pro Ala 420 425 430 AspSer Val Ala Ile Gly Arg Ser Arg Gln Val Val Lys Glu Gly Tyr 435 440 445Ala Lys Arg Leu Pro His His Pro Asp Gln Pro Gln 450 455 460 46 29 DNABacillus subtilis 46 gcggccgcgg tacctgtgtt acacctgtt 29 47 38 DNABacillus subtilis 47 gtcaagctta attctcatgt ttgacagctt atcatcgg 38 48 18DNA Bacillus subtilis 48 catgggagag acctttgg 18 49 17 DNA Bacillussubtilis 49 gtcggtcttc catttgc 17 50 17 DNA Bacillus subtilis 50cgaccactgt atcttgg 17 51 17 DNA Bacillus subtilis 51 gagatgccaa acagtgc17 52 16 DNA Bacillus subtilis 52 catgtccatc gtgacg 16 53 17 DNABacillus subtilis 53 caggagcatt tgatacg 17 54 16 DNA Bacillus subtilis54 ccttcagatg tgatcc 16 55 17 DNA Bacillus subtilis 55 gtgttgacgtcaactgc 17 56 18 DNA Bacillus subtilis 56 gttcagcctt tcctctcg 18 57 18DNA Bacillus subtilis 57 gctaccttct ttcttagg 18 58 18 DNA Bacillussubtilis 58 cgtcaatatg atctgtgc 18 59 17 DNA Bacillus subtilis 59ggaaagaagg tctgtgc 17 60 17 DNA Bacillus subtilis 60 cagctatcag ctgacag17 61 20 DNA Bacillus subtilis 61 gctcagctat gacatattcc 20 62 17 DNABacillus subtilis 62 gatcgtcttg attaccg 17 63 16 DNA Bacillus subtilis63 agctttatcg gtgacg 16 64 16 DNA Bacillus subtilis 64 tgagcacgat tgcagg16 65 17 DNA Bacillus subtilis 65 cattgcggag acattgc 17 66 26 DNABacillus subtilis 66 tagacaattg gaagagaaaa gagata 26 67 20 DNA Bacillussubtilis 67 ccgtcgctat tgtaaccagt 20 68 29 DNA Bacillus subtilis 68ggaattccaa agctgcagcg gccggcgcg 29 69 32 DNA Bacillus subtilis 69gaagatctcg tatacttggc ttctgcagct gc 32 70 31 DNA Bacillus subtilis 70gaagatctgg tcaacaagct ggaaagcact c 31 71 33 DNA Bacillus subtilis 71cccaagcttc gtgacgtaca gcaccgttcc ggc 33 72 50 DNA Bacillus subtilis 72ccttaagggc cgaatattta tacggagctc cctgaaacaa caaaaacggc 50 73 34 DNABacillus subtilis 73 ggtgttctct agagcggccg cggttgcggt cagc 34 74 27 DNABacillus subtilis 74 gtccttcttg gtacctggaa gcagagc 27 75 39 DNA Bacillussubtilis 75 gtataaatat tcggccctta aggccagtac cattttccc 39 76 13 DNABacillus subtilis 76 gggccggatc cgc 13 77 20 DNA Bacillus subtilis 77attcccggcc taggcgccgg 20 78 19 DNA Bacillus subtilis 78 ggaaattatcgtgatcaac 19 79 21 DNA Bacillus subtilis 79 gcacgagcac tgataaatat g 2180 21 DNA Bacillus subtilis 80 catatttatc agtgctcgtg c 21 81 17 DNABacillus subtilis 81 tcgtagacct catatgc 17 82 17 DNA Bacillus subtilis82 gtcgttaaac cgtgtgc 17 83 39 DNA Bacillus subtilis 83 ctagaggatccccgggtacc gtgctctgcc ttttagtcc 39 84 37 DNA Bacillus subtilis 84gtacatcgaa ttcgtgctca ttattaatct gttcagc 37 85 20 DNA Bacillus subtilis85 aactattgcc gatgataagc 20 86 1260 DNA Bacillus subtilis CDS(1)..(1260) 86 atg aaa aaa gtg atg tta gct acg gct ttg ttt tta gga ttgact cca 48 Met Lys Lys Val Met Leu Ala Thr Ala Leu Phe Leu Gly Leu ThrPro 1 5 10 15 gct ggc gcg aac gca gct gat tta ggc cac cag acg ttg ggatcc aat 96 Ala Gly Ala Asn Ala Ala Asp Leu Gly His Gln Thr Leu Gly SerAsn 20 25 30 gat ggc tgg ggc gcg tac tcg acc ggc acg aca ggc gga tca aaagca 144 Asp Gly Trp Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala35 40 45 tcc tcc tca aat gtg tat acc gtc agc aac aga aac cag ctt gtc tcg192 Ser Ser Ser Asn Val Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser 5055 60 gca tta ggg aag gaa acg aac aca acg cca aaa atc att tat atc aag240 Ala Leu Gly Lys Glu Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys 6570 75 80 gga acg att gac atg aac gtg gat gac aat ctg aag ccg ctt ggc cta288 Gly Thr Ile Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu 8590 95 aat gac tat aaa gat ccg gag tat gat ttg gac aaa tat ttg aaa gcc336 Asn Asp Tyr Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala 100105 110 tat gat cct agc aca tgg ggc aaa aaa gag ccg tcg gga aca caa gaa384 Tyr Asp Pro Ser Thr Trp Gly Lys Lys Glu Pro Ser Gly Thr Gln Glu 115120 125 gaa gcg aga gca cgc tct cag aaa aac caa aaa gca cgg gtc atg gtg432 Glu Ala Arg Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val 130135 140 gat atc cct gca aac acg acg atc gtc ggt tca ggg act aac gct aaa480 Asp Ile Pro Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys 145150 155 160 gtc gtg gga gga aac ttc caa atc aag agt gat aac gtc att attcgc 528 Val Val Gly Gly Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg165 170 175 aac att gaa ttc cag gat gcc tat gac tat ttt ccg caa tgg gatccg 576 Asn Ile Glu Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro180 185 190 act gac gga agc tca ggg aac tgg aac tca caa tac gac aac atcacg 624 Thr Asp Gly Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr195 200 205 ata aac ggc ggc aca cac atc tgg att gat cac tgt aca ttt aatgac 672 Ile Asn Gly Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp210 215 220 ggt tcg cgt ccg gac agc aca tca ccg aaa tat tat gga aga aaatat 720 Gly Ser Arg Pro Asp Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr225 230 235 240 cag cac cat gac ggc caa acg gat gct tcc aac ggt gct aactat atc 768 Gln His His Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn TyrIle 245 250 255 acg atg tcc tac aac tat tat cac gat cat gat aaa agc tccatt ttc 816 Thr Met Ser Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser IlePhe 260 265 270 gga tca agt gac agc aaa acc tcc gat gac ggc aaa tta aaaatt acg 864 Gly Ser Ser Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys IleThr 275 280 285 ctg cat cat aac cgc tat aaa aat att gtc cag cgc gcg ccgaga gtc 912 Leu His His Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala Pro ArgVal 290 295 300 cgc ttc ggg caa gtg cac gta tac aac aac tat tat gaa ggaagc aca 960 Arg Phe Gly Gln Val His Val Tyr Asn Asn Tyr Tyr Glu Gly SerThr 305 310 315 320 agc tct tca agt tat cct ttt agc tat gca tgg gga atcgga aag tca 1008 Ser Ser Ser Ser Tyr Pro Phe Ser Tyr Ala Trp Gly Ile GlyLys Ser 325 330 335 tct aaa atc tat gcc caa aac aat gtc att gac gta ccggga ctg tca 1056 Ser Lys Ile Tyr Ala Gln Asn Asn Val Ile Asp Val Pro GlyLeu Ser 340 345 350 gct gct aaa acg atc agc gta ttc agc ggg gga acg gcttta tat gac 1104 Ala Ala Lys Thr Ile Ser Val Phe Ser Gly Gly Thr Ala LeuTyr Asp 355 360 365 tcc ggc acg ttg ctg aac ggc aca cag atc aac gca tcggct gca aac 1152 Ser Gly Thr Leu Leu Asn Gly Thr Gln Ile Asn Ala Ser AlaAla Asn 370 375 380 ggg ctg agc tct tct gtc ggc tgg acg ccg tct ctg catgga tcg att 1200 Gly Leu Ser Ser Ser Val Gly Trp Thr Pro Ser Leu His GlySer Ile 385 390 395 400 gat gct tct gct aat gtg aaa tca aat gtt ata aatcaa gcg ggt gcg 1248 Asp Ala Ser Ala Asn Val Lys Ser Asn Val Ile Asn GlnAla Gly Ala 405 410 415 ggt aaa tta aat 1260 Gly Lys Leu Asn 420 87 420PRT Bacillus subtilis 87 Met Lys Lys Val Met Leu Ala Thr Ala Leu Phe LeuGly Leu Thr Pro 1 5 10 15 Ala Gly Ala Asn Ala Ala Asp Leu Gly His GlnThr Leu Gly Ser Asn 20 25 30 Asp Gly Trp Gly Ala Tyr Ser Thr Gly Thr ThrGly Gly Ser Lys Ala 35 40 45 Ser Ser Ser Asn Val Tyr Thr Val Ser Asn ArgAsn Gln Leu Val Ser 50 55 60 Ala Leu Gly Lys Glu Thr Asn Thr Thr Pro LysIle Ile Tyr Ile Lys 65 70 75 80 Gly Thr Ile Asp Met Asn Val Asp Asp AsnLeu Lys Pro Leu Gly Leu 85 90 95 Asn Asp Tyr Lys Asp Pro Glu Tyr Asp LeuAsp Lys Tyr Leu Lys Ala 100 105 110 Tyr Asp Pro Ser Thr Trp Gly Lys LysGlu Pro Ser Gly Thr Gln Glu 115 120 125 Glu Ala Arg Ala Arg Ser Gln LysAsn Gln Lys Ala Arg Val Met Val 130 135 140 Asp Ile Pro Ala Asn Thr ThrIle Val Gly Ser Gly Thr Asn Ala Lys 145 150 155 160 Val Val Gly Gly AsnPhe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg 165 170 175 Asn Ile Glu PheGln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro 180 185 190 Thr Asp GlySer Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr 195 200 205 Ile AsnGly Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp 210 215 220 GlySer Arg Pro Asp Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr 225 230 235240 Gln His His Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile 245250 255 Thr Met Ser Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe260 265 270 Gly Ser Ser Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys IleThr 275 280 285 Leu His His Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala ProArg Val 290 295 300 Arg Phe Gly Gln Val His Val Tyr Asn Asn Tyr Tyr GluGly Ser Thr 305 310 315 320 Ser Ser Ser Ser Tyr Pro Phe Ser Tyr Ala TrpGly Ile Gly Lys Ser 325 330 335 Ser Lys Ile Tyr Ala Gln Asn Asn Val IleAsp Val Pro Gly Leu Ser 340 345 350 Ala Ala Lys Thr Ile Ser Val Phe SerGly Gly Thr Ala Leu Tyr Asp 355 360 365 Ser Gly Thr Leu Leu Asn Gly ThrGln Ile Asn Ala Ser Ala Ala Asn 370 375 380 Gly Leu Ser Ser Ser Val GlyTrp Thr Pro Ser Leu His Gly Ser Ile 385 390 395 400 Asp Ala Ser Ala AsnVal Lys Ser Asn Val Ile Asn Gln Ala Gly Ala 405 410 415 Gly Lys Leu Asn420 88 26 DNA Bacillus subtilis 88 actagtaatg atggctgggg cgcgta 26 89 26DNA Bacillus subtilis 89 gtcgacatgt tgtcgtattg tgagtt 26 90 52 DNABacillus subtilis 90 gagctctaca acgcttatgg atccgcggcc gcggcggcacacacatctgg at 52 91 26 DNA Bacillus subtilis 91 gacgtcagcc cgtttgcagccgatgc 26 92 1257 DNA Streptococcus pyogenes CDS (1)..(1257) 92 gtg cctatt ttt aaa aaa act tta att gtt tta tcc ttt att ttt ttg 48 Val Pro IlePhe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu 1 5 10 15 ata tctatc ttg att tat cta aat atg tat cta ttt gga aca tca act 96 Ile Ser IleLeu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr 20 25 30 gta gga atttat gga gta ata tta ata acc tat cta gtt att aaa ctt 144 Val Gly Ile TyrGly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu 35 40 45 gga tta tct ttcctt tat gag cca ttt aaa gga aag cca cat gac tat 192 Gly Leu Ser Phe LeuTyr Glu Pro Phe Lys Gly Lys Pro His Asp Tyr 50 55 60 aaa gtt gct gct gtaatt cct tct tat aat gaa gat gcc gag tca tta 240 Lys Val Ala Ala Val IlePro Ser Tyr Asn Glu Asp Ala Glu Ser Leu 65 70 75 80 tta gaa act ctt aaaagt gtg tta gca cag acc tat ccg tta tca gaa 288 Leu Glu Thr Leu Lys SerVal Leu Ala Gln Thr Tyr Pro Leu Ser Glu 85 90 95 att tat att gtt gat gatggg agt tca aac aca gat gca ata caa tta 336 Ile Tyr Ile Val Asp Asp GlySer Ser Asn Thr Asp Ala Ile Gln Leu 100 105 110 att gaa gag tat gta aataga gaa gtg gat att tgt cga aac gtt atc 384 Ile Glu Glu Tyr Val Asn ArgGlu Val Asp Ile Cys Arg Asn Val Ile 115 120 125 gtt cac cgt tcc ctt gtcaat aaa gga aaa cgc cat gct caa gcg tgg 432 Val His Arg Ser Leu Val AsnLys Gly Lys Arg His Ala Gln Ala Trp 130 135 140 gca ttt gaa aga tct gacgct gac gtt ttt tta acc gta gat tca gat 480 Ala Phe Glu Arg Ser Asp AlaAsp Val Phe Leu Thr Val Asp Ser Asp 145 150 155 160 act tat atc tat ccaaat gcc tta gaa gaa ctc cta aaa agc ttc aat 528 Thr Tyr Ile Tyr Pro AsnAla Leu Glu Glu Leu Leu Lys Ser Phe Asn 165 170 175 gat gag aca gtt tatgct gca aca gga cat ttg aat gct aga aac aga 576 Asp Glu Thr Val Tyr AlaAla Thr Gly His Leu Asn Ala Arg Asn Arg 180 185 190 caa act aat cta ttaacg cga ctt aca gat atc cgt tac gat aat gcc 624 Gln Thr Asn Leu Leu ThrArg Leu Thr Asp Ile Arg Tyr Asp Asn Ala 195 200 205 ttt ggg gtg gag cgtgct gct caa tca tta aca ggt aat att tta gtt 672 Phe Gly Val Glu Arg AlaAla Gln Ser Leu Thr Gly Asn Ile Leu Val 210 215 220 tgc tca gga cca ttgagt att tat cga cgt gaa gtg att att cct aac 720 Cys Ser Gly Pro Leu SerIle Tyr Arg Arg Glu Val Ile Ile Pro Asn 225 230 235 240 tta gag cgc tataaa aat caa aca ttc cta ggt tta cct gtt agc att 768 Leu Glu Arg Tyr LysAsn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile 245 250 255 ggg gat gat cgatgt tta aca aat tat gct att gat tta gga cgc act 816 Gly Asp Asp Arg CysLeu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr 260 265 270 gtc tac caa tcaaca gct aga tgt gat act gat gta cct ttc caa tta 864 Val Tyr Gln Ser ThrAla Arg Cys Asp Thr Asp Val Pro Phe Gln Leu 275 280 285 aaa agt tat ttaaag caa caa aat cga tgg aat aaa tct ttt ttt aaa 912 Lys Ser Tyr Leu LysGln Gln Asn Arg Trp Asn Lys Ser Phe Phe Lys 290 295 300 gaa tct att atttct gtt aaa aaa att ctt tct aat ccc atc gtt gcc 960 Glu Ser Ile Ile SerVal Lys Lys Ile Leu Ser Asn Pro Ile Val Ala 305 310 315 320 tta tgg actatt ttc gaa gtc gtt atg ttt atg atg ttg att gtc gca 1008 Leu Trp Thr IlePhe Glu Val Val Met Phe Met Met Leu Ile Val Ala 325 330 335 att ggg aatctt ttg ttt aat caa gct att caa tta gac ctt att aaa 1056 Ile Gly Asn LeuLeu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys 340 345 350 ctt ttt gccttt tta tcc atc atc ttt atc gtt gct tta tgt cgt aat 1104 Leu Phe Ala PheLeu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360 365 gtt cat tatatg atc aaa cat cct gct agt ttt ttg tta tct cct ctg 1152 Val His Tyr MetIle Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu 370 375 380 tat gga atatta cac ttg ttt gtc tta cag ccc cta aaa ctt tat tct 1200 Tyr Gly Ile LeuHis Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser 385 390 395 400 tta tgcacc att aaa aat acg gaa tgg gga aca cgt aaa aag gtc act 1248 Leu Cys ThrIle Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr 405 410 415 att tttaaa 1257 Ile Phe Lys 93 419 PRT Streptococcus pyogenes 93 Val Pro IlePhe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu 1 5 10 15 Ile SerIle Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr 20 25 30 Val GlyIle Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu 35 40 45 Gly LeuSer Phe Leu Tyr Glu Pro Phe Lys Gly Lys Pro His Asp Tyr 50 55 60 Lys ValAla Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu 65 70 75 80 LeuGlu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu 85 90 95 IleTyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu 100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile 115 120125 Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp 130135 140 Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160 Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys SerPhe Asn 165 170 175 Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn AlaArg Asn Arg 180 185 190 Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile ArgTyr Asp Asn Ala 195 200 205 Phe Gly Val Glu Arg Ala Ala Gln Ser Leu ThrGly Asn Ile Leu Val 210 215 220 Cys Ser Gly Pro Leu Ser Ile Tyr Arg ArgGlu Val Ile Ile Pro Asn 225 230 235 240 Leu Glu Arg Tyr Lys Asn Gln ThrPhe Leu Gly Leu Pro Val Ser Ile 245 250 255 Gly Asp Asp Arg Cys Leu ThrAsn Tyr Ala Ile Asp Leu Gly Arg Thr 260 265 270 Val Tyr Gln Ser Thr AlaArg Cys Asp Thr Asp Val Pro Phe Gln Leu 275 280 285 Lys Ser Tyr Leu LysGln Gln Asn Arg Trp Asn Lys Ser Phe Phe Lys 290 295 300 Glu Ser Ile IleSer Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala 305 310 315 320 Leu TrpThr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala 325 330 335 IleGly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys 340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360365 Val His Tyr Met Ile Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu 370375 380 Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400 Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys LysVal Thr 405 410 415 Ile Phe Lys 94 2916 DNA Pasteurella multocida CDS(1)..(2916) 94 atg aat aca tta tca caa gca ata aaa gca tat aac agc aatgac tat 48 Met Asn Thr Leu Ser Gln Ala Ile Lys Ala Tyr Asn Ser Asn AspTyr 1 5 10 15 caa tta gca ctc aaa tta ttt gaa aag tcg gcg gaa atc tatgga cgg 96 Gln Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr GlyArg 20 25 30 aaa att gtt gaa ttt caa att acc aaa tgc caa gaa aaa ctc tcagca 144 Lys Ile Val Glu Phe Gln Ile Thr Lys Cys Gln Glu Lys Leu Ser Ala35 40 45 cat cct tct gtt aat tca gca cat ctt tct gta aat aaa gaa gaa aaa192 His Pro Ser Val Asn Ser Ala His Leu Ser Val Asn Lys Glu Glu Lys 5055 60 gtc aat gtt tgc gat agt ccg tta gat att gca aca caa ctg tta ctt240 Val Asn Val Cys Asp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu 6570 75 80 tcc aac gta aaa aaa tta gta ctt tct gac tcg gaa aaa aac acg tta288 Ser Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu 8590 95 aaa aat aaa tgg aaa ttg ctc act gag aag aaa tct gaa aat gcg gag336 Lys Asn Lys Trp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu 100105 110 gta aga gcg gtc gcc ctt gta cca aaa gat ttt ccc aaa gat ctg gtt384 Val Arg Ala Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val 115120 125 tta gcg cct tta cct gat cat gtt aat gat ttt aca tgg tac aaa aag432 Leu Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys 130135 140 cga aag aaa aga ctt ggc ata aaa cct gaa cat caa cat gtt ggt ctt480 Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu 145150 155 160 tct att atc gtt aca aca ttc aat cga cca gca att tta tcg attaca 528 Ser Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr165 170 175 tta gcc tgt tta gta aac caa aaa aca cat tac ccg ttt gaa gttatc 576 Leu Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu Val Ile180 185 190 gtg aca gat gat ggt agt cag gaa gat cta tca ccg atc att cgccaa 624 Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln195 200 205 tat gaa aat aaa ttg gat att cgc tac gtc aga caa aaa gat aacggt 672 Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly210 215 220 ttt caa gcc agt gcc gct cgg aat atg gga tta cgc tta gca aaatat 720 Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr225 230 235 240 gac ttt att ggc tta ctc gac tgt gat atg gcg cca aat ccatta tgg 768 Asp Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro LeuTrp 245 250 255 gtt cat tct tat gtt gca gag cta tta gaa gat gat gat ttaaca atc 816 Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu ThrIle 260 265 270 att ggt cca aga aaa tac atc gat aca caa cat att gac ccaaaa gac 864 Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro LysAsp 275 280 285 ttc tta aat aac gcg agt ttg ctt gaa tca tta cca gaa gtgaaa acc 912 Phe Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val LysThr 290 295 300 aat aat agt gtt gcc gca aaa ggg gaa gga aca gtt tct ctggat tgg 960 Asn Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu AspTrp 305 310 315 320 cgc tta gaa caa ttc gaa aaa aca gaa aat ctc cgc ttatcc gat tcg 1008 Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu SerAsp Ser 325 330 335 cct ttc cgt ttt ttt gcg gcg ggt aat gtt gct ttc gctaaa aaa tgg 1056 Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala LysLys Trp 340 345 350 cta aat aaa tcc ggt ttc ttt gat gag gaa ttt aat cactgg ggt gga 1104 Leu Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His TrpGly Gly 355 360 365 gaa gat gtg gaa ttt gga tat cgc tta ttc cgt tac ggtagt ttc ttt 1152 Glu Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly SerPhe Phe 370 375 380 aaa act att gat ggc att atg gcc tac cat caa gag ccacca ggt aaa 1200 Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro ProGly Lys 385 390 395 400 gaa aat gaa acc gat cgt gaa gcg gga aaa aat attacg ctc gat att 1248 Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile ThrLeu Asp Ile 405 410 415 atg aga gaa aag gtc cct tat atc tat aga aaa ctttta cca ata gaa 1296 Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu LeuPro Ile Glu 420 425 430 gat tcg cat atc aat aga gta cct tta gtt tca atttat atc cca gct 1344 Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile TyrIle Pro Ala 435 440 445 tat aac tgt gca aac tat att caa cgt tgc gta gatagt gca ctg aat 1392 Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp SerAla Leu Asn 450 455 460 cag act gtt gtt gat ctc gag gtt tgt att tgt aacgat ggt tca aca 1440 Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn AspGly Ser Thr 465 470 475 480 gat aat acc tta gaa gtg atc aat aag ctt tatggt aat aat cct agg 1488 Asp Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr GlyAsn Asn Pro Arg 485 490 495 gta cgc atc atg tct aaa cca aat ggc gga atagcc tca gca tca aat 1536 Val Arg Ile Met Ser Lys Pro Asn Gly Gly Ile AlaSer Ala Ser Asn 500 505 510 gca gcc gtt tct ttt gct aaa ggt tat tac attggg cag tta gat tca 1584 Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile GlyGln Leu Asp Ser 515 520 525 gat gat tat ctt gag cct gat gca gtt gaa ctgtgt tta aaa gaa ttt 1632 Asp Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu CysLeu Lys Glu Phe 530 535 540 tta aaa gat aaa acg cta gct tgt gtt tat accact aat aga aac gtc 1680 Leu Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr ThrAsn Arg Asn Val 545 550 555 560 aat ccg gat ggt agc tta atc gct aat ggttac aat tgg cca gaa ttt 1728 Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly TyrAsn Trp Pro Glu Phe 565 570 575 tca cga gaa aaa ctc aca acg gct atg attgct cac cac ttt aga atg 1776 Ser Arg Glu Lys Leu Thr Thr Ala Met Ile AlaHis His Phe Arg Met 580 585 590 ttc acg att aga gct tgg cat tta act gatgga ttc aat gaa aaa att 1824 Phe Thr Ile Arg Ala Trp His Leu Thr Asp GlyPhe Asn Glu Lys Ile 595 600 605 gaa aat gcc gta gac tat gac atg ttc ctcaaa ctc agt gaa gtt gga 1872 Glu Asn Ala Val Asp Tyr Asp Met Phe Leu LysLeu Ser Glu Val Gly 610 615 620 aaa ttt aaa cat ctt aat aaa atc tgc tataac cgt gta tta cat ggt 1920 Lys Phe Lys His Leu Asn Lys Ile Cys Tyr AsnArg Val Leu His Gly 625 630 635 640 gat aac aca tca att aag aaa ctt ggcatt caa aag aaa aac cat ttt 1968 Asp Asn Thr Ser Ile Lys Lys Leu Gly IleGln Lys Lys Asn His Phe 645 650 655 gtt gta gtc aat cag tca tta aat agacaa ggc ata act tat tat aat 2016 Val Val Val Asn Gln Ser Leu Asn Arg GlnGly Ile Thr Tyr Tyr Asn 660 665 670 tat gac gaa ttt gat gat tta gat gaaagt aga aag tat att ttc aat 2064 Tyr Asp Glu Phe Asp Asp Leu Asp Glu SerArg Lys Tyr Ile Phe Asn 675 680 685 aaa acc gct gaa tat caa gaa gag attgat atc tta aaa gat att aaa 2112 Lys Thr Ala Glu Tyr Gln Glu Glu Ile AspIle Leu Lys Asp Ile Lys 690 695 700 atc atc cag aat aaa gat gcc aaa atcgca gtc agt att ttt tat ccc 2160 Ile Ile Gln Asn Lys Asp Ala Lys Ile AlaVal Ser Ile Phe Tyr Pro 705 710 715 720 aat aca tta aac ggc tta gtg aaaaaa cta aac aat att att gaa tat 2208 Asn Thr Leu Asn Gly Leu Val Lys LysLeu Asn Asn Ile Ile Glu Tyr 725 730 735 aat aaa aat ata ttc gtt att gttcta cat gtt gat aag aat cat ctt 2256 Asn Lys Asn Ile Phe Val Ile Val LeuHis Val Asp Lys Asn His Leu 740 745 750 aca cca gat atc aaa aaa gaa atacta gcc ttc tat cat aaa cat caa 2304 Thr Pro Asp Ile Lys Lys Glu Ile LeuAla Phe Tyr His Lys His Gln 755 760 765 gtg aat att tta cta aat aat gatatc tca tat tac acg agt aat aga 2352 Val Asn Ile Leu Leu Asn Asn Asp IleSer Tyr Tyr Thr Ser Asn Arg 770 775 780 tta ata aaa act gag gcg cat ttaagt aat att aat aaa tta agt cag 2400 Leu Ile Lys Thr Glu Ala His Leu SerAsn Ile Asn Lys Leu Ser Gln 785 790 795 800 tta aat cta aat tgt gaa tacatc att ttt gat aat cat gac agc cta 2448 Leu Asn Leu Asn Cys Glu Tyr IleIle Phe Asp Asn His Asp Ser Leu 805 810 815 ttc gtt aaa aat gac agc tatgct tat atg aaa aaa tat gat gtc ggc 2496 Phe Val Lys Asn Asp Ser Tyr AlaTyr Met Lys Lys Tyr Asp Val Gly 820 825 830 atg aat ttc tca gca tta acacat gat tgg atc gag aaa atc aat gcg 2544 Met Asn Phe Ser Ala Leu Thr HisAsp Trp Ile Glu Lys Ile Asn Ala 835 840 845 cat cca cca ttt aaa aag ctcatt aaa act tat ttt aat gac aat gac 2592 His Pro Pro Phe Lys Lys Leu IleLys Thr Tyr Phe Asn Asp Asn Asp 850 855 860 tta aaa agt atg aat gtg aaaggg gca tca caa ggt atg ttt atg acg 2640 Leu Lys Ser Met Asn Val Lys GlyAla Ser Gln Gly Met Phe Met Thr 865 870 875 880 tat gcg cta gcg cat gagctt ctg acg att att aaa gaa gtc atc aca 2688 Tyr Ala Leu Ala His Glu LeuLeu Thr Ile Ile Lys Glu Val Ile Thr 885 890 895 tct tgc cag tca att gatagt gtg cca gaa tat aac act gag gat att 2736 Ser Cys Gln Ser Ile Asp SerVal Pro Glu Tyr Asn Thr Glu Asp Ile 900 905 910 tgg ttc caa ttt gca ctttta atc tta gaa aag aaa acc ggc cat gta 2784 Trp Phe Gln Phe Ala Leu LeuIle Leu Glu Lys Lys Thr Gly His Val 915 920 925 ttt aat aaa aca tcg accctg act tat atg cct tgg gaa cga aaa tta 2832 Phe Asn Lys Thr Ser Thr LeuThr Tyr Met Pro Trp Glu Arg Lys Leu 930 935 940 caa tgg aca aat gaa caaatt gaa agt gca aaa aga gga gaa aat ata 2880 Gln Trp Thr Asn Glu Gln IleGlu Ser Ala Lys Arg Gly Glu Asn Ile 945 950 955 960 cct gtt aac aag ttcatt att aat agt ata act cta 2916 Pro Val Asn Lys Phe Ile Ile Asn Ser IleThr Leu 965 970 95 972 PRT Pasteurella multocida 95 Met Asn Thr Leu SerGln Ala Ile Lys Ala Tyr Asn Ser Asn Asp Tyr 1 5 10 15 Gln Leu Ala LeuLys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly Arg 20 25 30 Lys Ile Val GluPhe Gln Ile Thr Lys Cys Gln Glu Lys Leu Ser Ala 35 40 45 His Pro Ser ValAsn Ser Ala His Leu Ser Val Asn Lys Glu Glu Lys 50 55 60 Val Asn Val CysAsp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu 65 70 75 80 Ser Asn ValLys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu 85 90 95 Lys Asn LysTrp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu 100 105 110 Val ArgAla Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val 115 120 125 LeuAla Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys 130 135 140Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu 145 150155 160 Ser Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr165 170 175 Leu Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu ValIle 180 185 190 Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile IleArg Gln 195 200 205 Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln LysAsp Asn Gly 210 215 220 Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu ArgLeu Ala Lys Tyr 225 230 235 240 Asp Phe Ile Gly Leu Leu Asp Cys Asp MetAla Pro Asn Pro Leu Trp 245 250 255 Val His Ser Tyr Val Ala Glu Leu LeuGlu Asp Asp Asp Leu Thr Ile 260 265 270 Ile Gly Pro Arg Lys Tyr Ile AspThr Gln His Ile Asp Pro Lys Asp 275 280 285 Phe Leu Asn Asn Ala Ser LeuLeu Glu Ser Leu Pro Glu Val Lys Thr 290 295 300 Asn Asn Ser Val Ala AlaLys Gly Glu Gly Thr Val Ser Leu Asp Trp 305 310 315 320 Arg Leu Glu GlnPhe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser 325 330 335 Pro Phe ArgPhe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp 340 345 350 Leu AsnLys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly 355 360 365 GluAsp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe 370 375 380Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys 385 390395 400 Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile405 410 415 Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro IleGlu 420 425 430 Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr IlePro Ala 435 440 445 Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp SerAla Leu Asn 450 455 460 Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys AsnAsp Gly Ser Thr 465 470 475 480 Asp Asn Thr Leu Glu Val Ile Asn Lys LeuTyr Gly Asn Asn Pro Arg 485 490 495 Val Arg Ile Met Ser Lys Pro Asn GlyGly Ile Ala Ser Ala Ser Asn 500 505 510 Ala Ala Val Ser Phe Ala Lys GlyTyr Tyr Ile Gly Gln Leu Asp Ser 515 520 525 Asp Asp Tyr Leu Glu Pro AspAla Val Glu Leu Cys Leu Lys Glu Phe 530 535 540 Leu Lys Asp Lys Thr LeuAla Cys Val Tyr Thr Thr Asn Arg Asn Val 545 550 555 560 Asn Pro Asp GlySer Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe 565 570 575 Ser Arg GluLys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met 580 585 590 Phe ThrIle Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile 595 600 605 GluAsn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly 610 615 620Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly 625 630635 640 Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe645 650 655 Val Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr TyrAsn 660 665 670 Tyr Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr IlePhe Asn 675 680 685 Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu LysAsp Ile Lys 690 695 700 Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val SerIle Phe Tyr Pro 705 710 715 720 Asn Thr Leu Asn Gly Leu Val Lys Lys LeuAsn Asn Ile Ile Glu Tyr 725 730 735 Asn Lys Asn Ile Phe Val Ile Val LeuHis Val Asp Lys Asn His Leu 740 745 750 Thr Pro Asp Ile Lys Lys Glu IleLeu Ala Phe Tyr His Lys His Gln 755 760 765 Val Asn Ile Leu Leu Asn AsnAsp Ile Ser Tyr Tyr Thr Ser Asn Arg 770 775 780 Leu Ile Lys Thr Glu AlaHis Leu Ser Asn Ile Asn Lys Leu Ser Gln 785 790 795 800 Leu Asn Leu AsnCys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu 805 810 815 Phe Val LysAsn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly 820 825 830 Met AsnPhe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala 835 840 845 HisPro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp 850 855 860Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr 865 870875 880 Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr885 890 895 Ser Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu AspIle 900 905 910 Trp Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr GlyHis Val 915 920 925 Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp GluArg Lys Leu 930 935 940 Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys ArgGly Glu Asn Ile 945 950 955 960 Pro Val Asn Lys Phe Ile Ile Asn Ser IleThr Leu 965 970 96 1206 DNA Streptococcus pyogenes CDS (1)..(1206) 96atg aaa ata gca gtt gct gga tca gga tat gtt gga tta tca cta gga 48 MetLys Ile Ala Val Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Gly 1 5 10 15gtt ctt tta tca ctt caa aac gaa gtc act att gtt gat att ctt ccc 96 ValLeu Leu Ser Leu Gln Asn Glu Val Thr Ile Val Asp Ile Leu Pro 20 25 30 tctaaa gtt gat aag att aat aat ggc tta tca cca att caa gat gaa 144 Ser LysVal Asp Lys Ile Asn Asn Gly Leu Ser Pro Ile Gln Asp Glu 35 40 45 tat attgaa tat tac tta aaa agt aag caa tta tct att aaa gca act 192 Tyr Ile GluTyr Tyr Leu Lys Ser Lys Gln Leu Ser Ile Lys Ala Thr 50 55 60 tta gat agcaaa gca gct tat aaa gaa gcg gaa ctg gtc att att gcc 240 Leu Asp Ser LysAla Ala Tyr Lys Glu Ala Glu Leu Val Ile Ile Ala 65 70 75 80 aca cct acaaat tac aac agt aga att aat tat ttt gat aca cag cat 288 Thr Pro Thr AsnTyr Asn Ser Arg Ile Asn Tyr Phe Asp Thr Gln His 85 90 95 gtt gaa aca gttatc aaa gag gta cta agc gtt aat agc cat gca act 336 Val Glu Thr Val IleLys Glu Val Leu Ser Val Asn Ser His Ala Thr 100 105 110 ctt atc atc aaatca aca att cca ata ggt ttc att act gaa atg aga 384 Leu Ile Ile Lys SerThr Ile Pro Ile Gly Phe Ile Thr Glu Met Arg 115 120 125 cag aaa ttc caaact gat cgt att atc ttc agc cct gaa ttt tta aga 432 Gln Lys Phe Gln ThrAsp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130 135 140 gaa tct aaa gcttta tat gac aac tta tat cca agc cga att att gtt 480 Glu Ser Lys Ala LeuTyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145 150 155 160 tct tgt gaagaa aac gat tct cca aaa gta aag gca gac gca gaa aaa 528 Ser Cys Glu GluAsn Asp Ser Pro Lys Val Lys Ala Asp Ala Glu Lys 165 170 175 ttt gca ctttta tta aag tct gca gct aaa aaa aat aat gta cca gta 576 Phe Ala Leu LeuLeu Lys Ser Ala Ala Lys Lys Asn Asn Val Pro Val 180 185 190 ctt att atggga gct tca gaa gct gaa gca gta aaa cta ttt gcc aat 624 Leu Ile Met GlyAla Ser Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195 200 205 act tat ttagcg tta agg gta gct tat ttt aat gag tta gac act tac 672 Thr Tyr Leu AlaLeu Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr Tyr 210 215 220 gca gaa tcgaga aaa tta aat agt cac atg att att caa gga att tct 720 Ala Glu Ser ArgLys Leu Asn Ser His Met Ile Ile Gln Gly Ile Ser 225 230 235 240 tat gatgat cga ata gga atg cat tat aat aac cca tca ttt ggt tat 768 Tyr Asp AspArg Ile Gly Met His Tyr Asn Asn Pro Ser Phe Gly Tyr 245 250 255 gga ggttat tgt cta cct aaa gat acg aag caa tta ttg gca aat tac 816 Gly Gly TyrCys Leu Pro Lys Asp Thr Lys Gln Leu Leu Ala Asn Tyr 260 265 270 aat aatatt cct caa acg cta att gaa gct atc gtt tca tca aat aat 864 Asn Asn IlePro Gln Thr Leu Ile Glu Ala Ile Val Ser Ser Asn Asn 275 280 285 gtg cgcaag tcc tat att gct aag caa att atc aac gtc tta gaa gag 912 Val Arg LysSer Tyr Ile Ala Lys Gln Ile Ile Asn Val Leu Glu Glu 290 295 300 cgg gagtcc cca gta aaa gta gtc ggg gtt tac cgt tta att atg aaa 960 Arg Glu SerPro Val Lys Val Val Gly Val Tyr Arg Leu Ile Met Lys 305 310 315 320 agtaac tca gat aat ttt aga gaa agt gct atc aaa gat gtt att gac 1008 Ser AsnSer Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Val Ile Asp 325 330 335 attctt aaa agt aaa gac att aag ata att att tat gag cca atg tta 1056 Ile LeuLys Ser Lys Asp Ile Lys Ile Ile Ile Tyr Glu Pro Met Leu 340 345 350 aacaaa ctt gaa tct gaa gat caa tct gta ctt gta aat gat tta gag 1104 Asn LysLeu Glu Ser Glu Asp Gln Ser Val Leu Val Asn Asp Leu Glu 355 360 365 aatttc aag aaa caa gca aat att atc gta act aat cgc tat gat aat 1152 Asn PheLys Lys Gln Ala Asn Ile Ile Val Thr Asn Arg Tyr Asp Asn 370 375 380 gaatta caa gat gtt aaa aat aaa gtt tac agt aga gat att ttt aat 1200 Glu LeuGln Asp Val Lys Asn Lys Val Tyr Ser Arg Asp Ile Phe Asn 385 390 395 400aga gac 1206 Arg Asp 97 402 PRT Streptococcus pyogenes 97 Met Lys IleAla Val Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Gly 1 5 10 15 Val LeuLeu Ser Leu Gln Asn Glu Val Thr Ile Val Asp Ile Leu Pro 20 25 30 Ser LysVal Asp Lys Ile Asn Asn Gly Leu Ser Pro Ile Gln Asp Glu 35 40 45 Tyr IleGlu Tyr Tyr Leu Lys Ser Lys Gln Leu Ser Ile Lys Ala Thr 50 55 60 Leu AspSer Lys Ala Ala Tyr Lys Glu Ala Glu Leu Val Ile Ile Ala 65 70 75 80 ThrPro Thr Asn Tyr Asn Ser Arg Ile Asn Tyr Phe Asp Thr Gln His 85 90 95 ValGlu Thr Val Ile Lys Glu Val Leu Ser Val Asn Ser His Ala Thr 100 105 110Leu Ile Ile Lys Ser Thr Ile Pro Ile Gly Phe Ile Thr Glu Met Arg 115 120125 Gln Lys Phe Gln Thr Asp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130135 140 Glu Ser Lys Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val145 150 155 160 Ser Cys Glu Glu Asn Asp Ser Pro Lys Val Lys Ala Asp AlaGlu Lys 165 170 175 Phe Ala Leu Leu Leu Lys Ser Ala Ala Lys Lys Asn AsnVal Pro Val 180 185 190 Leu Ile Met Gly Ala Ser Glu Ala Glu Ala Val LysLeu Phe Ala Asn 195 200 205 Thr Tyr Leu Ala Leu Arg Val Ala Tyr Phe AsnGlu Leu Asp Thr Tyr 210 215 220 Ala Glu Ser Arg Lys Leu Asn Ser His MetIle Ile Gln Gly Ile Ser 225 230 235 240 Tyr Asp Asp Arg Ile Gly Met HisTyr Asn Asn Pro Ser Phe Gly Tyr 245 250 255 Gly Gly Tyr Cys Leu Pro LysAsp Thr Lys Gln Leu Leu Ala Asn Tyr 260 265 270 Asn Asn Ile Pro Gln ThrLeu Ile Glu Ala Ile Val Ser Ser Asn Asn 275 280 285 Val Arg Lys Ser TyrIle Ala Lys Gln Ile Ile Asn Val Leu Glu Glu 290 295 300 Arg Glu Ser ProVal Lys Val Val Gly Val Tyr Arg Leu Ile Met Lys 305 310 315 320 Ser AsnSer Asp Asn Phe Arg Glu Ser Ala Ile Lys Asp Val Ile Asp 325 330 335 IleLeu Lys Ser Lys Asp Ile Lys Ile Ile Ile Tyr Glu Pro Met Leu 340 345 350Asn Lys Leu Glu Ser Glu Asp Gln Ser Val Leu Val Asn Asp Leu Glu 355 360365 Asn Phe Lys Lys Gln Ala Asn Ile Ile Val Thr Asn Arg Tyr Asp Asn 370375 380 Glu Leu Gln Asp Val Lys Asn Lys Val Tyr Ser Arg Asp Ile Phe Asn385 390 395 400 Arg Asp 98 912 DNA Streptococcus pyogenes CDS (1)..(912)98 atg acc aaa gtc aga aaa gcc att att cct gct gca ggt cta gga aca 48Met Thr Lys Val Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr 1 5 1015 cgt ttt tta cct gct acc aaa gct ctt gcc aaa gag atg ttg ccc atc 96Arg Phe Leu Pro Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro Ile 20 25 30gtt gat aaa cca acc atc cag ttt atc gtc gaa gaa gcg cta aaa tct 144 ValAsp Lys Pro Thr Ile Gln Phe Ile Val Glu Glu Ala Leu Lys Ser 35 40 45 ggcatc gag gaa atc ctt gtg gtg acc gga aaa gct aaa cgc tct atc 192 Gly IleGlu Glu Ile Leu Val Val Thr Gly Lys Ala Lys Arg Ser Ile 50 55 60 gag gaccat ttt gat tca aac ttt gaa tta gaa tac aac ctc caa gct 240 Glu Asp HisPhe Asp Ser Asn Phe Glu Leu Glu Tyr Asn Leu Gln Ala 65 70 75 80 aag gggaaa aat gaa ctg ttg aaa tta gtg gat gaa acc act gcc att 288 Lys Gly LysAsn Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ala Ile 85 90 95 aac ctt catttt atc cgt caa agc cac cca aga ggg ctg gga gat gct 336 Asn Leu His PheIle Arg Gln Ser His Pro Arg Gly Leu Gly Asp Ala 100 105 110 gtc tta caagcc aaa gcc ttt gtg ggc aat gaa ccc ttt gtg gtc atg 384 Val Leu Gln AlaLys Ala Phe Val Gly Asn Glu Pro Phe Val Val Met 115 120 125 ctt gga gatgac tta atg gac att aca aat gca tcc gct aaa cct ctc 432 Leu Gly Asp AspLeu Met Asp Ile Thr Asn Ala Ser Ala Lys Pro Leu 130 135 140 acc aaa caactc atg gag gac tat gac aag acg cat gca tcc act atc 480 Thr Lys Gln LeuMet Glu Asp Tyr Asp Lys Thr His Ala Ser Thr Ile 145 150 155 160 gct gtgatg aaa gtt cct cat gaa gat gtg tct agc tat ggg gtt atc 528 Ala Val MetLys Val Pro His Glu Asp Val Ser Ser Tyr Gly Val Ile 165 170 175 gct cctcaa ggc aag gct gtc aag ggc ctt tac agt gta gac acc ttt 576 Ala Pro GlnGly Lys Ala Val Lys Gly Leu Tyr Ser Val Asp Thr Phe 180 185 190 gtt gaaaaa cca caa cca gaa gat gcg cct agt gat ttg gct att att 624 Val Glu LysPro Gln Pro Glu Asp Ala Pro Ser Asp Leu Ala Ile Ile 195 200 205 ggt cgttac ctc cta acc cct gaa att ttt ggt att ttg gaa aga cag 672 Gly Arg TyrLeu Leu Thr Pro Glu Ile Phe Gly Ile Leu Glu Arg Gln 210 215 220 acc cctgga gca ggt aac gaa gtg caa ctc aca gat gct atc gat acc 720 Thr Pro GlyAla Gly Asn Glu Val Gln Leu Thr Asp Ala Ile Asp Thr 225 230 235 240 ctcaat aaa act cag cgt gtc ttt gca cga gaa ttt aaa ggc aat cgt 768 Leu AsnLys Thr Gln Arg Val Phe Ala Arg Glu Phe Lys Gly Asn Arg 245 250 255 tacgat gtt ggg gat aaa ttt gga ttc atg aaa aca tct atc gac tat 816 Tyr AspVal Gly Asp Lys Phe Gly Phe Met Lys Thr Ser Ile Asp Tyr 260 265 270 gcctta gaa cac cca cag gtc aaa gag gac ttg aaa aat tac att atc 864 Ala LeuGlu His Pro Gln Val Lys Glu Asp Leu Lys Asn Tyr Ile Ile 275 280 285 aaacta gga aaa gct ttg gaa aaa agt aaa gta cca aca cat tca aag 912 Lys LeuGly Lys Ala Leu Glu Lys Ser Lys Val Pro Thr His Ser Lys 290 295 300 99304 PRT Streptococcus pyogenes 99 Met Thr Lys Val Arg Lys Ala Ile IlePro Ala Ala Gly Leu Gly Thr 1 5 10 15 Arg Phe Leu Pro Ala Thr Lys AlaLeu Ala Lys Glu Met Leu Pro Ile 20 25 30 Val Asp Lys Pro Thr Ile Gln PheIle Val Glu Glu Ala Leu Lys Ser 35 40 45 Gly Ile Glu Glu Ile Leu Val ValThr Gly Lys Ala Lys Arg Ser Ile 50 55 60 Glu Asp His Phe Asp Ser Asn PheGlu Leu Glu Tyr Asn Leu Gln Ala 65 70 75 80 Lys Gly Lys Asn Glu Leu LeuLys Leu Val Asp Glu Thr Thr Ala Ile 85 90 95 Asn Leu His Phe Ile Arg GlnSer His Pro Arg Gly Leu Gly Asp Ala 100 105 110 Val Leu Gln Ala Lys AlaPhe Val Gly Asn Glu Pro Phe Val Val Met 115 120 125 Leu Gly Asp Asp LeuMet Asp Ile Thr Asn Ala Ser Ala Lys Pro Leu 130 135 140 Thr Lys Gln LeuMet Glu Asp Tyr Asp Lys Thr His Ala Ser Thr Ile 145 150 155 160 Ala ValMet Lys Val Pro His Glu Asp Val Ser Ser Tyr Gly Val Ile 165 170 175 AlaPro Gln Gly Lys Ala Val Lys Gly Leu Tyr Ser Val Asp Thr Phe 180 185 190Val Glu Lys Pro Gln Pro Glu Asp Ala Pro Ser Asp Leu Ala Ile Ile 195 200205 Gly Arg Tyr Leu Leu Thr Pro Glu Ile Phe Gly Ile Leu Glu Arg Gln 210215 220 Thr Pro Gly Ala Gly Asn Glu Val Gln Leu Thr Asp Ala Ile Asp Thr225 230 235 240 Leu Asn Lys Thr Gln Arg Val Phe Ala Arg Glu Phe Lys GlyAsn Arg 245 250 255 Tyr Asp Val Gly Asp Lys Phe Gly Phe Met Lys Thr SerIle Asp Tyr 260 265 270 Ala Leu Glu His Pro Gln Val Lys Glu Asp Leu LysAsn Tyr Ile Ile 275 280 285 Lys Leu Gly Lys Ala Leu Glu Lys Ser Lys ValPro Thr His Ser Lys 290 295 300 100 1347 DNA Streptococcus equizooepidemicus CDS (1)..(1347) 100 atg tca cat att aca ttt gat tat tcaaag gtt ctt gag caa ttt gcc 48 Met Ser His Ile Thr Phe Asp Tyr Ser LysVal Leu Glu Gln Phe Ala 1 5 10 15 gga cag cat gaa att gac ttt tta caaggt cag gta aca gag gct gat 96 Gly Gln His Glu Ile Asp Phe Leu Gln GlyGln Val Thr Glu Ala Asp 20 25 30 cag gca cta cgt cag ggc act gga cct ggatca gat ttc ttg ggc tgg 144 Gln Ala Leu Arg Gln Gly Thr Gly Pro Gly SerAsp Phe Leu Gly Trp 35 40 45 ctt gag tta cct gaa aac tat gac aaa gaa gaattt gct cgt atc ctt 192 Leu Glu Leu Pro Glu Asn Tyr Asp Lys Glu Glu PheAla Arg Ile Leu 50 55 60 aaa gca gct gag aag att aag gct gac agt gac gttctt gtt gtg att 240 Lys Ala Ala Glu Lys Ile Lys Ala Asp Ser Asp Val LeuVal Val Ile 65 70 75 80 ggt att ggt ggc tct tac ctt ggt gct aag gct gcaatt gac ttt ttg 288 Gly Ile Gly Gly Ser Tyr Leu Gly Ala Lys Ala Ala IleAsp Phe Leu 85 90 95 aac agc cat ttt gcc aac cta caa aca gca aaa gag cgcaaa gca cca 336 Asn Ser His Phe Ala Asn Leu Gln Thr Ala Lys Glu Arg LysAla Pro 100 105 110 caa att ctt tat gct ggt aac tcc atc tca tca agc tatctt gct gat 384 Gln Ile Leu Tyr Ala Gly Asn Ser Ile Ser Ser Ser Tyr LeuAla Asp 115 120 125 ctt gtg gac tat gtt caa gat aaa gat ttc tct gtt aacgtg att tct 432 Leu Val Asp Tyr Val Gln Asp Lys Asp Phe Ser Val Asn ValIle Ser 130 135 140 aag tct ggt aca aca aca gag cct gca atc gcc ttt cgtgtc ttt aaa 480 Lys Ser Gly Thr Thr Thr Glu Pro Ala Ile Ala Phe Arg ValPhe Lys 145 150 155 160 gaa tta ctt gtt aaa aag tac ggt caa gaa gag gccaac aag cgt atc 528 Glu Leu Leu Val Lys Lys Tyr Gly Gln Glu Glu Ala AsnLys Arg Ile 165 170 175 tat gca acg act gat aag gtc aag ggt gct gtt aaggtt gag gct gat 576 Tyr Ala Thr Thr Asp Lys Val Lys Gly Ala Val Lys ValGlu Ala Asp 180 185 190 gca aat cat tgg gaa acc ttt gtt gtg cca gat aatgtt ggt ggc cgt 624 Ala Asn His Trp Glu Thr Phe Val Val Pro Asp Asn ValGly Gly Arg 195 200 205 ttc tca gtg ctg aca gct gtg ggc ttg cta cca attgca gca tca ggg 672 Phe Ser Val Leu Thr Ala Val Gly Leu Leu Pro Ile AlaAla Ser Gly 210 215 220 gct gat att acc gcg ctg atg gaa gga gca aat gcagct cgt aag gac 720 Ala Asp Ile Thr Ala Leu Met Glu Gly Ala Asn Ala AlaArg Lys Asp 225 230 235 240 ctg tca tca gat aaa atc tca gaa aac atc gcttac caa tat gct gtg 768 Leu Ser Ser Asp Lys Ile Ser Glu Asn Ile Ala TyrGln Tyr Ala Val 245 250 255 gtc cgc aat atc ctc tat cgc aaa ggc tat gtaact gaa att ttg gca 816 Val Arg Asn Ile Leu Tyr Arg Lys Gly Tyr Val ThrGlu Ile Leu Ala 260 265 270 aac tat gag cca tca ttg cag tat ttt agc gaatgg tgg aag caa ctg 864 Asn Tyr Glu Pro Ser Leu Gln Tyr Phe Ser Glu TrpTrp Lys Gln Leu 275 280 285 gct ggt gag tct gaa gga aag gac caa aag ggtatt tac cca act tca 912 Ala Gly Glu Ser Glu Gly Lys Asp Gln Lys Gly IleTyr Pro Thr Ser 290 295 300 gct aat ttc tcg aca gac ctg cat tct ctt ggtcaa ttt atc caa gaa 960 Ala Asn Phe Ser Thr Asp Leu His Ser Leu Gly GlnPhe Ile Gln Glu 305 310 315 320 ggc tac cgt aac ctc ttt gag aca gtg attcgt gtg gac aag cca cgt 1008 Gly Tyr Arg Asn Leu Phe Glu Thr Val Ile ArgVal Asp Lys Pro Arg 325 330 335 caa aat gtg att atc cca gaa atg gct gaggac ctt gat ggc ctt ggc 1056 Gln Asn Val Ile Ile Pro Glu Met Ala Glu AspLeu Asp Gly Leu Gly 340 345 350 tac cta caa gga aaa gac gtt gac ttt gtcaac aaa aaa gca aca gat 1104 Tyr Leu Gln Gly Lys Asp Val Asp Phe Val AsnLys Lys Ala Thr Asp 355 360 365 ggt gtc ctt ctt gcc cat aca gat ggt ggtgtg cca aat atg ttt atc 1152 Gly Val Leu Leu Ala His Thr Asp Gly Gly ValPro Asn Met Phe Ile 370 375 380 acg ctt cca gag caa gac gaa ttt aca ctaggc tat acg atc tac ttc 1200 Thr Leu Pro Glu Gln Asp Glu Phe Thr Leu GlyTyr Thr Ile Tyr Phe 385 390 395 400 ttt gag ctt gct att gcc ctt tca ggctac ctc aac ggg gtc aat cca 1248 Phe Glu Leu Ala Ile Ala Leu Ser Gly TyrLeu Asn Gly Val Asn Pro 405 410 415 ttt gat cag cca ggc gtt gag gct tacaag aaa aac atg ttt gcc ctt 1296 Phe Asp Gln Pro Gly Val Glu Ala Tyr LysLys Asn Met Phe Ala Leu 420 425 430 ctt ggt aag cca ggc ttt gaa gag ctagga gca gcg ctc aac gca cgc 1344 Leu Gly Lys Pro Gly Phe Glu Glu Leu GlyAla Ala Leu Asn Ala Arg 435 440 445 ttg 1347 Leu 101 449 PRTStreptococcus equi zooepidemicus 101 Met Ser His Ile Thr Phe Asp Tyr SerLys Val Leu Glu Gln Phe Ala 1 5 10 15 Gly Gln His Glu Ile Asp Phe LeuGln Gly Gln Val Thr Glu Ala Asp 20 25 30 Gln Ala Leu Arg Gln Gly Thr GlyPro Gly Ser Asp Phe Leu Gly Trp 35 40 45 Leu Glu Leu Pro Glu Asn Tyr AspLys Glu Glu Phe Ala Arg Ile Leu 50 55 60 Lys Ala Ala Glu Lys Ile Lys AlaAsp Ser Asp Val Leu Val Val Ile 65 70 75 80 Gly Ile Gly Gly Ser Tyr LeuGly Ala Lys Ala Ala Ile Asp Phe Leu 85 90 95 Asn Ser His Phe Ala Asn LeuGln Thr Ala Lys Glu Arg Lys Ala Pro 100 105 110 Gln Ile Leu Tyr Ala GlyAsn Ser Ile Ser Ser Ser Tyr Leu Ala Asp 115 120 125 Leu Val Asp Tyr ValGln Asp Lys Asp Phe Ser Val Asn Val Ile Ser 130 135 140 Lys Ser Gly ThrThr Thr Glu Pro Ala Ile Ala Phe Arg Val Phe Lys 145 150 155 160 Glu LeuLeu Val Lys Lys Tyr Gly Gln Glu Glu Ala Asn Lys Arg Ile 165 170 175 TyrAla Thr Thr Asp Lys Val Lys Gly Ala Val Lys Val Glu Ala Asp 180 185 190Ala Asn His Trp Glu Thr Phe Val Val Pro Asp Asn Val Gly Gly Arg 195 200205 Phe Ser Val Leu Thr Ala Val Gly Leu Leu Pro Ile Ala Ala Ser Gly 210215 220 Ala Asp Ile Thr Ala Leu Met Glu Gly Ala Asn Ala Ala Arg Lys Asp225 230 235 240 Leu Ser Ser Asp Lys Ile Ser Glu Asn Ile Ala Tyr Gln TyrAla Val 245 250 255 Val Arg Asn Ile Leu Tyr Arg Lys Gly Tyr Val Thr GluIle Leu Ala 260 265 270 Asn Tyr Glu Pro Ser Leu Gln Tyr Phe Ser Glu TrpTrp Lys Gln Leu 275 280 285 Ala Gly Glu Ser Glu Gly Lys Asp Gln Lys GlyIle Tyr Pro Thr Ser 290 295 300 Ala Asn Phe Ser Thr Asp Leu His Ser LeuGly Gln Phe Ile Gln Glu 305 310 315 320 Gly Tyr Arg Asn Leu Phe Glu ThrVal Ile Arg Val Asp Lys Pro Arg 325 330 335 Gln Asn Val Ile Ile Pro GluMet Ala Glu Asp Leu Asp Gly Leu Gly 340 345 350 Tyr Leu Gln Gly Lys AspVal Asp Phe Val Asn Lys Lys Ala Thr Asp 355 360 365 Gly Val Leu Leu AlaHis Thr Asp Gly Gly Val Pro Asn Met Phe Ile 370 375 380 Thr Leu Pro GluGln Asp Glu Phe Thr Leu Gly Tyr Thr Ile Tyr Phe 385 390 395 400 Phe GluLeu Ala Ile Ala Leu Ser Gly Tyr Leu Asn Gly Val Asn Pro 405 410 415 PheAsp Gln Pro Gly Val Glu Ala Tyr Lys Lys Asn Met Phe Ala Leu 420 425 430Leu Gly Lys Pro Gly Phe Glu Glu Leu Gly Ala Ala Leu Asn Ala Arg 435 440445 Leu 102 1251 DNA Streptococcus uberis CDS (1)..(1251) 102 atg gaaaaa cta aaa aat ctc att aca ttt atg act ttt att ttc ctg 48 Met Glu LysLeu Lys Asn Leu Ile Thr Phe Met Thr Phe Ile Phe Leu 1 5 10 15 tgg ctcata att att ggg ctt aat gtt ttt gta ttt gga act aaa gga 96 Trp Leu IleIle Ile Gly Leu Asn Val Phe Val Phe Gly Thr Lys Gly 20 25 30 agt cta acagtg tat ggg att att cta tta acc tat ttg tcg ata aaa 144 Ser Leu Thr ValTyr Gly Ile Ile Leu Leu Thr Tyr Leu Ser Ile Lys 35 40 45 atg gga tta tctttt ttt tat cgt ccc tat aaa gga agt gta ggt caa 192 Met Gly Leu Ser PhePhe Tyr Arg Pro Tyr Lys Gly Ser Val Gly Gln 50 55 60 tat aag gta gca gctatt atc cca tct tat aat gag gat ggt gtc ggt 240 Tyr Lys Val Ala Ala IleIle Pro Ser Tyr Asn Glu Asp Gly Val Gly 65 70 75 80 tta cta gaa act ctaaag agt gtt caa aaa caa aca tat cca att gca 288 Leu Leu Glu Thr Leu LysSer Val Gln Lys Gln Thr Tyr Pro Ile Ala 85 90 95 gaa att ttc gta att gacgat ggg tca gta gat aaa aca ggt ata aaa 336 Glu Ile Phe Val Ile Asp AspGly Ser Val Asp Lys Thr Gly Ile Lys 100 105 110 ttg gtc gaa gac tat gtgaag tta aat ggc ttt gga gac caa gtt atc 384 Leu Val Glu Asp Tyr Val LysLeu Asn Gly Phe Gly Asp Gln Val Ile 115 120 125 gtt cat cag atg cct gaaaat gtt ggt aaa aga cat gct cag gct tgg 432 Val His Gln Met Pro Glu AsnVal Gly Lys Arg His Ala Gln Ala Trp 130 135 140 gca ttt gaa agg tct gatgct gat gtt ttc tta aca gtg gat tca gat 480 Ala Phe Glu Arg Ser Asp AlaAsp Val Phe Leu Thr Val Asp Ser Asp 145 150 155 160 acc tac atc tat cctgat gct ctt gaa gaa tta tta aag aca ttt aat 528 Thr Tyr Ile Tyr Pro AspAla Leu Glu Glu Leu Leu Lys Thr Phe Asn 165 170 175 gat cca gag gtc tacgct gca act ggt cat tta aat gca aga aat aga 576 Asp Pro Glu Val Tyr AlaAla Thr Gly His Leu Asn Ala Arg Asn Arg 180 185 190 caa act aat ctc ttaact aga ctg act gat att cgt tac gat aat gca 624 Gln Thr Asn Leu Leu ThrArg Leu Thr Asp Ile Arg Tyr Asp Asn Ala 195 200 205 ttt ggt gta gaa cgtgct gct cag tct gtt acg gga aat att ttg gtt 672 Phe Gly Val Glu Arg AlaAla Gln Ser Val Thr Gly Asn Ile Leu Val 210 215 220 tgt tcc gga cct ttaagt att tat aga cgt tcc gtc ggt att cca aat 720 Cys Ser Gly Pro Leu SerIle Tyr Arg Arg Ser Val Gly Ile Pro Asn 225 230 235 240 ctt gaa cgc tatacc tca caa aca ttt ctt ggt gtc cct gta agc ata 768 Leu Glu Arg Tyr ThrSer Gln Thr Phe Leu Gly Val Pro Val Ser Ile 245 250 255 ggg gat gac cgttgt ttg aca aat tat gca act gat ttg gga aaa acg 816 Gly Asp Asp Arg CysLeu Thr Asn Tyr Ala Thr Asp Leu Gly Lys Thr 260 265 270 gtt tat cag tcaact gca aga tgt gat act gac gtt cca gat aag ttt 864 Val Tyr Gln Ser ThrAla Arg Cys Asp Thr Asp Val Pro Asp Lys Phe 275 280 285 aag gtt ttc atcaaa caa caa aat cgt tgg aat aag tca ttt ttt agg 912 Lys Val Phe Ile LysGln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg 290 295 300 gag tct att atctct gtt aag aag tta tta gcc aca cca agt gtt gct 960 Glu Ser Ile Ile SerVal Lys Lys Leu Leu Ala Thr Pro Ser Val Ala 305 310 315 320 gtt tgg actatt aca gaa gtt tcc atg ttc atc atg cta gtt tat tct 1008 Val Trp Thr IleThr Glu Val Ser Met Phe Ile Met Leu Val Tyr Ser 325 330 335 atc ttt agctta ttg ata gga gag gct caa gaa ttt aat ctc ata aaa 1056 Ile Phe Ser LeuLeu Ile Gly Glu Ala Gln Glu Phe Asn Leu Ile Lys 340 345 350 ctg gtt gctttt tta gtt att att ttc ata gta gct ctt tgt aga aat 1104 Leu Val Ala PheLeu Val Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360 365 gtt cat tacatg gtt aag cat cca ttt gct ttt tta ttg tca ccg ttt 1152 Val His Tyr MetVal Lys His Pro Phe Ala Phe Leu Leu Ser Pro Phe 370 375 380 tat gga ttgata cat cta ttc gtt ttg caa cct ctt aag ata tat tcg 1200 Tyr Gly Leu IleHis Leu Phe Val Leu Gln Pro Leu Lys Ile Tyr Ser 385 390 395 400 tta tttact ata aga aat gct aca tgg gga act cgt aaa aag aca agt 1248 Leu Phe ThrIle Arg Asn Ala Thr Trp Gly Thr Arg Lys Lys Thr Ser 405 410 415 aaa 1251Lys 103 417 PRT Streptococcus uberis 103 Met Glu Lys Leu Lys Asn Leu IleThr Phe Met Thr Phe Ile Phe Leu 1 5 10 15 Trp Leu Ile Ile Ile Gly LeuAsn Val Phe Val Phe Gly Thr Lys Gly 20 25 30 Ser Leu Thr Val Tyr Gly IleIle Leu Leu Thr Tyr Leu Ser Ile Lys 35 40 45 Met Gly Leu Ser Phe Phe TyrArg Pro Tyr Lys Gly Ser Val Gly Gln 50 55 60 Tyr Lys Val Ala Ala Ile IlePro Ser Tyr Asn Glu Asp Gly Val Gly 65 70 75 80 Leu Leu Glu Thr Leu LysSer Val Gln Lys Gln Thr Tyr Pro Ile Ala 85 90 95 Glu Ile Phe Val Ile AspAsp Gly Ser Val Asp Lys Thr Gly Ile Lys 100 105 110 Leu Val Glu Asp TyrVal Lys Leu Asn Gly Phe Gly Asp Gln Val Ile 115 120 125 Val His Gln MetPro Glu Asn Val Gly Lys Arg His Ala Gln Ala Trp 130 135 140 Ala Phe GluArg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp 145 150 155 160 ThrTyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe Asn 165 170 175Asp Pro Glu Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg 180 185190 Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala 195200 205 Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu Val210 215 220 Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Ser Val Gly Ile ProAsn 225 230 235 240 Leu Glu Arg Tyr Thr Ser Gln Thr Phe Leu Gly Val ProVal Ser Ile 245 250 255 Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr AspLeu Gly Lys Thr 260 265 270 Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr AspVal Pro Asp Lys Phe 275 280 285 Lys Val Phe Ile Lys Gln Gln Asn Arg TrpAsn Lys Ser Phe Phe Arg 290 295 300 Glu Ser Ile Ile Ser Val Lys Lys LeuLeu Ala Thr Pro Ser Val Ala 305 310 315 320 Val Trp Thr Ile Thr Glu ValSer Met Phe Ile Met Leu Val Tyr Ser 325 330 335 Ile Phe Ser Leu Leu IleGly Glu Ala Gln Glu Phe Asn Leu Ile Lys 340 345 350 Leu Val Ala Phe LeuVal Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360 365 Val His Tyr MetVal Lys His Pro Phe Ala Phe Leu Leu Ser Pro Phe 370 375 380 Tyr Gly LeuIle His Leu Phe Val Leu Gln Pro Leu Lys Ile Tyr Ser 385 390 395 400 LeuPhe Thr Ile Arg Asn Ala Thr Trp Gly Thr Arg Lys Lys Thr Ser 405 410 415Lys 104 1203 DNA Streptococcus uberis CDS (1)..(1203) 104 gtg aaa attgca gtt gca ggt tct ggc tat gtt ggc cta tca tta agt 48 Val Lys Ile AlaVal Ala Gly Ser Gly Tyr Val Gly Leu Ser Leu Ser 1 5 10 15 gta tta ttagca cag aaa aat cct gtt aca gtt gta gat att att gag 96 Val Leu Leu AlaGln Lys Asn Pro Val Thr Val Val Asp Ile Ile Glu 20 25 30 aag aaa gta aatctc ata aat caa aaa caa tca cca atc cag gat gtt 144 Lys Lys Val Asn LeuIle Asn Gln Lys Gln Ser Pro Ile Gln Asp Val 35 40 45 gat att gaa aac tattta aaa gaa aaa aag tta caa tta aga gct act 192 Asp Ile Glu Asn Tyr LeuLys Glu Lys Lys Leu Gln Leu Arg Ala Thr 50 55 60 cta gac gcc gat caa gcattt agg gat gca gat ata cta att att gct 240 Leu Asp Ala Asp Gln Ala PheArg Asp Ala Asp Ile Leu Ile Ile Ala 65 70 75 80 aca cca acc aat tat gatgtg gag aag aat ttt ttt gat act agt cat 288 Thr Pro Thr Asn Tyr Asp ValGlu Lys Asn Phe Phe Asp Thr Ser His 85 90 95 gtt gag act gta att gag aaagct tta gct tta aat agt cag gct ttg 336 Val Glu Thr Val Ile Glu Lys AlaLeu Ala Leu Asn Ser Gln Ala Leu 100 105 110 tta gtt att aaa tca acg atacca ctt ggt ttt att aaa aag atg cgt 384 Leu Val Ile Lys Ser Thr Ile ProLeu Gly Phe Ile Lys Lys Met Arg 115 120 125 caa aaa tat cag aca gac cgtatt att ttt agt ccc gaa ttt ctt aga 432 Gln Lys Tyr Gln Thr Asp Arg IleIle Phe Ser Pro Glu Phe Leu Arg 130 135 140 gag tct aaa gct tta aaa gataat ctt tat cct agt cga ata att gtt 480 Glu Ser Lys Ala Leu Lys Asp AsnLeu Tyr Pro Ser Arg Ile Ile Val 145 150 155 160 tcc ttt gaa gat gat gattct atg gaa gta ata gaa gca gca aag act 528 Ser Phe Glu Asp Asp Asp SerMet Glu Val Ile Glu Ala Ala Lys Thr 165 170 175 ttt gct caa ttg tta aaagat ggt tct ttg gat aaa gat gtt cct gta 576 Phe Ala Gln Leu Leu Lys AspGly Ser Leu Asp Lys Asp Val Pro Val 180 185 190 ctt ttt atg ggt tca gcagag gct gaa gca gta aaa tta ttt gcc aat 624 Leu Phe Met Gly Ser Ala GluAla Glu Ala Val Lys Leu Phe Ala Asn 195 200 205 acc tat tta gct atg cgtgtc tcc tat ttt aat gag tta gat aca tat 672 Thr Tyr Leu Ala Met Arg ValSer Tyr Phe Asn Glu Leu Asp Thr Tyr 210 215 220 gct gaa aag aat ggt ttacgt gtg gat aat att att gag ggc gtt tgc 720 Ala Glu Lys Asn Gly Leu ArgVal Asp Asn Ile Ile Glu Gly Val Cys 225 230 235 240 cat gat cga cgc atagga att cat tat aat aac cct tct ttt ggc tat 768 His Asp Arg Arg Ile GlyIle His Tyr Asn Asn Pro Ser Phe Gly Tyr 245 250 255 gga gga tac tgc ttacct aaa gat acc aaa cag ttg cta gca ggc tat 816 Gly Gly Tyr Cys Leu ProLys Asp Thr Lys Gln Leu Leu Ala Gly Tyr 260 265 270 gat ggt att cct caatcg ctt ata aaa gca att gtt gat tct aat aaa 864 Asp Gly Ile Pro Gln SerLeu Ile Lys Ala Ile Val Asp Ser Asn Lys 275 280 285 att cgt aaa gag tatatc gca tca caa att tta caa caa ttg agt gat 912 Ile Arg Lys Glu Tyr IleAla Ser Gln Ile Leu Gln Gln Leu Ser Asp 290 295 300 att aat gta gat cctaaa gat gca acg att ggt att tac cgc ctt atc 960 Ile Asn Val Asp Pro LysAsp Ala Thr Ile Gly Ile Tyr Arg Leu Ile 305 310 315 320 atg aaa agt aactct gat aat ttc aga gag agt gca ata aaa gat att 1008 Met Lys Ser Asn SerAsp Asn Phe Arg Glu Ser Ala Ile Lys Asp Ile 325 330 335 att gat cat attaag agc tat caa att aat ata gtc ttg tat gag cca 1056 Ile Asp His Ile LysSer Tyr Gln Ile Asn Ile Val Leu Tyr Glu Pro 340 345 350 atg atg aat gaagat ttt gat tta cca atc att gat gat tta tct gac 1104 Met Met Asn Glu AspPhe Asp Leu Pro Ile Ile Asp Asp Leu Ser Asp 355 360 365 ttc aaa gcc atgtca cat att atc gtt tca aat aga tat gat tta gcc 1152 Phe Lys Ala Met SerHis Ile Ile Val Ser Asn Arg Tyr Asp Leu Ala 370 375 380 tta gaa gat gttaaa gaa aaa gtt tac acc aga gat att tac ggt gtg 1200 Leu Glu Asp Val LysGlu Lys Val Tyr Thr Arg Asp Ile Tyr Gly Val 385 390 395 400 gat 1203 Asp105 401 PRT Streptococcus uberis 105 Val Lys Ile Ala Val Ala Gly Ser GlyTyr Val Gly Leu Ser Leu Ser 1 5 10 15 Val Leu Leu Ala Gln Lys Asn ProVal Thr Val Val Asp Ile Ile Glu 20 25 30 Lys Lys Val Asn Leu Ile Asn GlnLys Gln Ser Pro Ile Gln Asp Val 35 40 45 Asp Ile Glu Asn Tyr Leu Lys GluLys Lys Leu Gln Leu Arg Ala Thr 50 55 60 Leu Asp Ala Asp Gln Ala Phe ArgAsp Ala Asp Ile Leu Ile Ile Ala 65 70 75 80 Thr Pro Thr Asn Tyr Asp ValGlu Lys Asn Phe Phe Asp Thr Ser His 85 90 95 Val Glu Thr Val Ile Glu LysAla Leu Ala Leu Asn Ser Gln Ala Leu 100 105 110 Leu Val Ile Lys Ser ThrIle Pro Leu Gly Phe Ile Lys Lys Met Arg 115 120 125 Gln Lys Tyr Gln ThrAsp Arg Ile Ile Phe Ser Pro Glu Phe Leu Arg 130 135 140 Glu Ser Lys AlaLeu Lys Asp Asn Leu Tyr Pro Ser Arg Ile Ile Val 145 150 155 160 Ser PheGlu Asp Asp Asp Ser Met Glu Val Ile Glu Ala Ala Lys Thr 165 170 175 PheAla Gln Leu Leu Lys Asp Gly Ser Leu Asp Lys Asp Val Pro Val 180 185 190Leu Phe Met Gly Ser Ala Glu Ala Glu Ala Val Lys Leu Phe Ala Asn 195 200205 Thr Tyr Leu Ala Met Arg Val Ser Tyr Phe Asn Glu Leu Asp Thr Tyr 210215 220 Ala Glu Lys Asn Gly Leu Arg Val Asp Asn Ile Ile Glu Gly Val Cys225 230 235 240 His Asp Arg Arg Ile Gly Ile His Tyr Asn Asn Pro Ser PheGly Tyr 245 250 255 Gly Gly Tyr Cys Leu Pro Lys Asp Thr Lys Gln Leu LeuAla Gly Tyr 260 265 270 Asp Gly Ile Pro Gln Ser Leu Ile Lys Ala Ile ValAsp Ser Asn Lys 275 280 285 Ile Arg Lys Glu Tyr Ile Ala Ser Gln Ile LeuGln Gln Leu Ser Asp 290 295 300 Ile Asn Val Asp Pro Lys Asp Ala Thr IleGly Ile Tyr Arg Leu Ile 305 310 315 320 Met Lys Ser Asn Ser Asp Asn PheArg Glu Ser Ala Ile Lys Asp Ile 325 330 335 Ile Asp His Ile Lys Ser TyrGln Ile Asn Ile Val Leu Tyr Glu Pro 340 345 350 Met Met Asn Glu Asp PheAsp Leu Pro Ile Ile Asp Asp Leu Ser Asp 355 360 365 Phe Lys Ala Met SerHis Ile Ile Val Ser Asn Arg Tyr Asp Leu Ala 370 375 380 Leu Glu Asp ValLys Glu Lys Val Tyr Thr Arg Asp Ile Tyr Gly Val 385 390 395 400 Asp 106912 DNA Streptococcus uberis CDS (1)..(912) 106 atg act aaa gta aga aaagcc att att cca gct gcc gga ctt ggc aca 48 Met Thr Lys Val Arg Lys AlaIle Ile Pro Ala Ala Gly Leu Gly Thr 1 5 10 15 cgt ttt tta cca gca acaaaa gct ctc gct aag gaa atg ttg ccc atc 96 Arg Phe Leu Pro Ala Thr LysAla Leu Ala Lys Glu Met Leu Pro Ile 20 25 30 gtt gac aaa cca acc att caattc atc gtg gaa gaa gct ttg cgt tct 144 Val Asp Lys Pro Thr Ile Gln PheIle Val Glu Glu Ala Leu Arg Ser 35 40 45 ggc att gaa gaa atc ttg gtc gtaaca gga aaa tca aaa cgc tcc att 192 Gly Ile Glu Glu Ile Leu Val Val ThrGly Lys Ser Lys Arg Ser Ile 50 55 60 gaa gac cat ttt gat tcc aac ttt gaactc gaa tat aat ttg caa gaa 240 Glu Asp His Phe Asp Ser Asn Phe Glu LeuGlu Tyr Asn Leu Gln Glu 65 70 75 80 aaa ggg aaa act gaa ctc tta aaa ttagtt gat gaa acc act tct ata 288 Lys Gly Lys Thr Glu Leu Leu Lys Leu ValAsp Glu Thr Thr Ser Ile 85 90 95 aac ttg cat ttc att cgt caa agt cat cccaaa ggc tta ggg gat gct 336 Asn Leu His Phe Ile Arg Gln Ser His Pro LysGly Leu Gly Asp Ala 100 105 110 gtt tta caa gca aaa gct ttt gta gga aatgaa ccc ttc att gtt atg 384 Val Leu Gln Ala Lys Ala Phe Val Gly Asn GluPro Phe Ile Val Met 115 120 125 ctt ggt gac gat ttg atg gac att aca aatacc aaa gct gtc cca tta 432 Leu Gly Asp Asp Leu Met Asp Ile Thr Asn ThrLys Ala Val Pro Leu 130 135 140 acc aaa caa tta atg gac gat tat gaa acaaca cat gct tct aca ata 480 Thr Lys Gln Leu Met Asp Asp Tyr Glu Thr ThrHis Ala Ser Thr Ile 145 150 155 160 gcc gta atg aaa gtt cct cac gat gacgta tcc tct tat ggt gtc att 528 Ala Val Met Lys Val Pro His Asp Asp ValSer Ser Tyr Gly Val Ile 165 170 175 gct cca aac ggc aaa gcc ttg aat ggctta tat agc gtg gat acc ttt 576 Ala Pro Asn Gly Lys Ala Leu Asn Gly LeuTyr Ser Val Asp Thr Phe 180 185 190 gtt gaa aaa cca aaa cct gag gac gcacca agt gac ctt gct atc att 624 Val Glu Lys Pro Lys Pro Glu Asp Ala ProSer Asp Leu Ala Ile Ile 195 200 205 gga cgt tat ctc tta aca cct gaa attttt gac att ctt gaa aat caa 672 Gly Arg Tyr Leu Leu Thr Pro Glu Ile PheAsp Ile Leu Glu Asn Gln 210 215 220 gca cca ggt gcc gga aac gaa gtc caatta act gat gct atc gat acc 720 Ala Pro Gly Ala Gly Asn Glu Val Gln LeuThr Asp Ala Ile Asp Thr 225 230 235 240 ctc aac aaa aca caa cgt gtt tttgct cgt gag ttt act ggc aaa cgc 768 Leu Asn Lys Thr Gln Arg Val Phe AlaArg Glu Phe Thr Gly Lys Arg 245 250 255 tac gat gtt gga gac aag ttt ggcttc atg aaa aca tct atc gat tat 816 Tyr Asp Val Gly Asp Lys Phe Gly PheMet Lys Thr Ser Ile Asp Tyr 260 265 270 gcc cta aaa cac cat caa gtc aaagat gac cta aaa gct tat att atc 864 Ala Leu Lys His His Gln Val Lys AspAsp Leu Lys Ala Tyr Ile Ile 275 280 285 aag tta ggt aaa gaa tta gaa aaagca caa gat tcc aaa gaa agc aaa 912 Lys Leu Gly Lys Glu Leu Glu Lys AlaGln Asp Ser Lys Glu Ser Lys 290 295 300 107 304 PRT Streptococcus uberis107 Met Thr Lys Val Arg Lys Ala Ile Ile Pro Ala Ala Gly Leu Gly Thr 1 510 15 Arg Phe Leu Pro Ala Thr Lys Ala Leu Ala Lys Glu Met Leu Pro Ile 2025 30 Val Asp Lys Pro Thr Ile Gln Phe Ile Val Glu Glu Ala Leu Arg Ser 3540 45 Gly Ile Glu Glu Ile Leu Val Val Thr Gly Lys Ser Lys Arg Ser Ile 5055 60 Glu Asp His Phe Asp Ser Asn Phe Glu Leu Glu Tyr Asn Leu Gln Glu 6570 75 80 Lys Gly Lys Thr Glu Leu Leu Lys Leu Val Asp Glu Thr Thr Ser Ile85 90 95 Asn Leu His Phe Ile Arg Gln Ser His Pro Lys Gly Leu Gly Asp Ala100 105 110 Val Leu Gln Ala Lys Ala Phe Val Gly Asn Glu Pro Phe Ile ValMet 115 120 125 Leu Gly Asp Asp Leu Met Asp Ile Thr Asn Thr Lys Ala ValPro Leu 130 135 140 Thr Lys Gln Leu Met Asp Asp Tyr Glu Thr Thr His AlaSer Thr Ile 145 150 155 160 Ala Val Met Lys Val Pro His Asp Asp Val SerSer Tyr Gly Val Ile 165 170 175 Ala Pro Asn Gly Lys Ala Leu Asn Gly LeuTyr Ser Val Asp Thr Phe 180 185 190 Val Glu Lys Pro Lys Pro Glu Asp AlaPro Ser Asp Leu Ala Ile Ile 195 200 205 Gly Arg Tyr Leu Leu Thr Pro GluIle Phe Asp Ile Leu Glu Asn Gln 210 215 220 Ala Pro Gly Ala Gly Asn GluVal Gln Leu Thr Asp Ala Ile Asp Thr 225 230 235 240 Leu Asn Lys Thr GlnArg Val Phe Ala Arg Glu Phe Thr Gly Lys Arg 245 250 255 Tyr Asp Val GlyAsp Lys Phe Gly Phe Met Lys Thr Ser Ile Asp Tyr 260 265 270 Ala Leu LysHis His Gln Val Lys Asp Asp Leu Lys Ala Tyr Ile Ile 275 280 285 Lys LeuGly Lys Glu Leu Glu Lys Ala Gln Asp Ser Lys Glu Ser Lys 290 295 300 1085158 DNA Streptococcus equisimilis 108 tcaatttatg gctttttgct gatagcttacctattagtca aaatgtcctt atcctttttt 60 tacaagccat ttaagggaag ggctgggcaatataaggttg cagccattat tccctcttat 120 aacgaagatg ctgagtcatt gctagagaccttaaaaagtg ttcagcagca aacctatccc 180 ctagcagaaa tttatgttgt tgacgatggaagtgctgatg agacaggtat taagcgcatt 240 gaagactatg tgcgtgacac tggtgacctatcaagcaatg tcattgttca tcggtcagag 300 aaaaatcaag gaaagcgtca tgcacaggcctgggcctttg aaagatcaga cgctgatgtc 360 tttttgaccg ttgactcaga tacttatatctaccctgatg ctttagagga gttgttaaaa 420 acctttaatg acccaactgt ttttgctgcgacgggtcacc ttaatgtcag aaatagacaa 480 accaatctct taacacgctt gacagatattcgctatgata atgcttttgg cgttgaacga 540 gctgcccaat ccgttacagg taatatccttgtttgctcag gtccgcttag cgtttacaga 600 cgcgaggtgg ttgttcctaa catagatagatacatcaacc agaccttcct gggtattcct 660 gtaagtattg gtgatgacag gtgcttgaccaactatgcaa ctgatttagg aaagactgtt 720 tatcaatcca ctgctaaatg tattacagatgttcctgaca agatgtctac ttacttgaag 780 cagcaaaacc gctggaacaa gtccttctttagagagtcca ttatttctgt taagaaaatc 840 atgaacaatc cttttgtagc cctatggaccatacttgagg tgtctatgtt tatgatgctt 900 gtttattctg tggtggattt ctttgtaggcaatgtcagag aatttgattg gctcagggtt 960 ttagcctttc tggtgattat cttcattgttgccctgtgtc ggaacattca ttacatgctt 1020 aagcacccgc tgtccttctt gttatctccgttttatgggg tgctgcattt gtttgtccta 1080 cagcccttga aattatattc tctttttactattagaaatg ctgactgggg aacacgtaaa 1140 aaattattat aaaccaacta gacctaggttctgacaaggg agctaagcta gggataaaca 1200 aagagttttg atccgactcg agcagctcataaacgaaagc tatcccactt gtaattgaag 1260 ctaagagctt ttagcttgca gctctataaagacgaaccag aggctgagtg tcagctttgg 1320 tgtgagggct aggtcattat gatccttcaggtgtggcacc tgagctccgg cagtagctaa 1380 ctgtactaag gtatcaaagg aaaaaatgaagtgaaaattt ctgtagcagg ctcaggatat 1440 gtcggcctat ccttgagtat tttactggcacaacataatg acgtcactgt tgttgacatt 1500 attgatgaaa aggtgagatt gatcaatcaaggcatatcgc caatcaagga tgctgatatt 1560 gaggagtatt taaaaaatgc gccgctaaatctcacagcga cgcttgatgg cgcaagcgct 1620 tatagcaatg cagaccttat tatcattgctactccgacaa attatgacag cgaacgcaac 1680 tactttgaca caaggcatgt tgaagaggtcatcgagcagg tcctagacct aaatgcgtca 1740 gcaaccatta ttatcaaatc aaccataccactaggcttta tcaagcatgt tagggaaaaa 1800 taccagacag atcgtattat ttttagcccagaatttttaa gagaatcaaa agccttatac 1860 gataaccttt acccaagtcg gatcattgtttcttatgaaa aggacgactc accaagggtt 1920 attcaggctg ctaaagcctt tgctggtcttttaaaggaag gagccaaaag caaggatact 1980 ccggtcttat ttatgggctc acaggaggctgaggcggtca agctatttgc gaataccttt 2040 ttggctatgc gggtgtctta ctttaatgaattagacacct attccgaaag caagggtcta 2100 gatgctcagc gcgtgattga aggagtctgtcatgatcagc gcattggtaa ccattacaat 2160 aacccttcct ttggatatgg cggctattgcctgccaaagg acagcaagca gctgttggca 2220 aattatagag gcattcccca gtccttgatgtcagcgattg ttgaatccaa caagatacga 2280 aaatcttatt tggctgaaca aatattagacagagcctcta gtcaaaagca ggctggtgta 2340 ccattaacga ttggctttta ccgcttgattatgaaaagca actctgataa tttccgagaa 2400 agcgccatta aagatattat tgatatcatcaacgactatg gggttaatat tgtcatttac 2460 gaacccatgc ttggcgagga tattggctacagggttgtca aggacttaga gcagttcaaa 2520 aacgagtcta caatcattgt gtcaaatcgctttgaggacg acctaggaga tgtcattgat 2580 aaggtttata cgagagatgt ctttggaagagactagtcag aaaacgaatg gcactcataa 2640 ggaaccacaa atcaaggagg aactcatgacaaaggtcaga aaagccatta tcccagccgc 2700 cggcctaggc actcgcttcc tgcccgccaccaaggcactg gccaaggaaa tgctcccaat 2760 cgtcgataag ccaaccattc aattcatcgtcgaggaagcc ctaaaggcag gtatcgagga 2820 gattcttgtc gtcaccggca aggccaaacgctctatcgag gaccactttg actccaactt 2880 cgagctcgaa tacaatctcc aagccaagggcaaaaccgag ctactcaagc tcgttgatga 2940 gaccactgcc atcaacctgc acttcattcgtcagagccac cctagaggac taggggacgc 3000 tgtcctccaa gccaaggcct ttgttggcaatgagcccttt gtggtcatgc tgggggatga 3060 cctcatggat attaccaatc ctagtgccaagcccttgacc aagcagctta ttgaggatta 3120 tgattgcaca cacgcctcaa cgattgcagtgatgagggtg ccgcatgagg aggtttccaa 3180 ttatggtgtg attgcaccgc aagggaaggctgttaagggc ttgtatagtg tggagacctt 3240 tgttgagaag ccaagtccag atgaggcaccgagtgactta gcgattattg gtcgatattt 3300 gttgacgcct gagatttttg ccatattggagaagcaggcg cctggagctg gcaatgaggt 3360 acagctgacc gatgcgattg acaagctcaataagacacag cgggtttttg cgagggagtt 3420 taagggagag cggtatgatg ttggggacaagtttggcttt atgaagacct cacttgacta 3480 tgctctcaag caccctcagg tcaaggacgacctcactgac tacattataa agctcagtaa 3540 gcaactgaac aaggacgtca agaaataggcgtttattgat cagctattgc agagctattt 3600 aaaagcattt agagctttaa ggtgggatactagaggattg gtatctcact ttttaggctg 3660 acttgtatta ataccaaaag ccaaaactaggcagataagc ataaggaatt agattaaaaa 3720 taaggaacca aaacatgaaa aactacgccattatcctagc agctggaaag ggaacgcgca 3780 tgaagtcagc gcttcccaag gtgctgcacaaggtatcagg cctaagcatg ctggagcatg 3840 tcctcaagag tgtctcagcc ctagcccctcaaaagcagct cacagtgatc ggtcatcagg 3900 cagagcaggt gcgtgctgtc ctaggagagcaatcgctaac agtggtgcaa gaggagcagc 3960 tagggacagg ccatgcagtc atgatggcagaagaggagct atctggctta gaggggcaaa 4020 ccctagtgat tgcaggtgac acccccttgatcagaggaga aagcctcaag gctctgctag 4080 actatcatat cagagaaaag aatgtggcaaccattctcac agccaatgcc aaggatccct 4140 ttggctatgg acgaatcatt cgcaatgcagcaggagaggt ggtcaacatc gttgagcaaa 4200 aggatgctaa tgaggcagag caagaggtcaaggagatcaa cacagggact tatatctttg 4260 acaataagcg cctttttgag gctctaaagcatctcacgac tgataatgcc caaggggagt 4320 actacctaac cgatgtgatc agtattttcaaggctggcca agaaagggtt ggcgcttacc 4380 tgctgaagga ctttgatgag agcctaggggttaatgatcg cttagctcta gcccaggccg 4440 aggtgattat gcaagagcgg atcaacaggcagcacatgct taatggggtg accctgcaaa 4500 acccggcagc tacctatatt gaaagcagtgtagagattgc accagacgtc ttgattgaag 4560 ccaatgtgac cttaaaggga cagactagaattggcagcag aagtgtcata agcaatggga 4620 gctatatcct tgattcgagg cttggtgagggtgtagtggt tagccagtcg gtgattgagg 4680 cttcagtctt agcagatgga gtgacagtagggccatatgc acacattcgc ccggactccc 4740 agctcgatga gtgtgttcat attgggaactttgtagaggt taaggggtct catctagggg 4800 ccaataccaa ggcagggcat ttgacttacctggggaatgc cgagattggc tcagaggtta 4860 acattggtgc aggaagcatt acggttaattatgatggtca acggaaatac cagacagtga 4920 ttggcgatca cgcttttatt gggagtcattcgactttgat agctccggta gaggttgggg 4980 agaatgcttt aacagcagca gggtctacgatagcccagtc agtgccggca gacagtgtgg 5040 ctatagggcg cagccgtcag gtggtgaaggaaggctatgc caagaggctg ccgcaccacc 5100 caaatcaagc ctaatcgctc aaccaaaagaggcaggtgag aaaacctagg ccattaaa 5158

What is claimed is:
 1. A method for producing a hyaluronic acid,comprising: (a) cultivating a Bacillus host cell under conditionssuitable for production of the hyaluronic acid, wherein the Bacillushost cell comprises a nucleic acid construct comprising a hyaluronansynthase encoding sequence operably linked to a promoter sequenceforeign to the hyaluronan synthase encoding sequence; and (b) recoveringthe hyaluronic acid from the cultivation medium.
 2. The method of claim1, wherein the hyaluronan synthase encoding sequence encodes a Group Ihyaluronan synthase.
 3. The method of claim 2, wherein the Group Ihyaluronan synthase encoding sequence is obtained from a Streptococcusstrain.
 4. The method of claim 3, wherein the Streptococcus strain isStreptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis,or Streptococcus equi subsp. zooepidemicus.
 5. The method of claim 1,wherein the hyaluronan synthase encoding sequence is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 2, SEQ IDNO: 93, or SEQ ID NO: 103; (b) a nucleic acid sequence which hybridizesunder low, medium, or high stringency conditions with SEQ ID NO: 1, SEQID NO: 92, or SEQ ID NO: 102; and (c) a complementary strand of (a) or(b).
 6. The method of claim 5, wherein the hyaluronan synthase encodingsequence encodes a polypeptide having the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; or a fragment thereof havinghyaluronan synthase activity.
 7. The method of claim 1, wherein thehyaluronan synthase encoding sequence encodes a Group II hyaluronansynthase.
 8. The method of claim 7, wherein the Group II hyaluronansynthase encoding sequence is obtained from a Pasteurella strain.
 9. Themethod of claim 8, wherein the Pasteurella strain is Pasteurellamultocida.
 10. The method of claim 1, wherein the hyaluronan synthaseencoding sequence is selected from the group consisting of (a) a nucleicacid sequence encoding a polypeptide with an amino acid sequence havingat least about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 95; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 94; and (c) a complementary strand of (a) or (b).
 11. The method ofclaim 10, wherein the hyaluronan synthase encoding sequence encodes apolypeptide having the amino acid sequence of SEQ ID NO: 95, or afragment thereof having hyaluronan synthase activity.
 12. The method ofclaim 1, wherein a precursor sugar of the hyaluronic acid is supplied toor produced by the Bacillus host cell.
 13. The method of claim 12,wherein the precursor sugar is D-glucuronic acid orN-acetyl-glucosamine.
 14. The method of claim 12, wherein the precursorsugar is encoded by endogenous genes, by non-endogenous genes, or by acombination of endogenous and non-endogenous genes in the Bacillus hostcell.
 15. The method of claim 1, wherein the nucleic acid constructfurther comprises one or more genes encoding enzymes in the biosynthesisof a precursor sugar of the hyaluronic acid or the Bacillus host cellfurther comprises one or more second nucleic acid constructs comprisingone or more genes encoding enzymes in the biosynthesis of a precursorsugar of the hyaluronic acid.
 16. The method of claim 15, wherein theone or more genes is selected from the group consisting of a UDP-glucose6-dehydrogenase gene, UDP-glucose pyrophosphorylase gene,UDP-N-acetylglucosamine pyrophosphorylase gene, glucose-6-phosphateisomerase gene, hexokinase gene, phosphoglucomutase gene,amidotransferase gene, mutase gene, and acetyl transferase gene.
 17. Themethod of claim 16, wherein the UDP-glucose 6-dehydrogenase gene is ahasB gene or tuaD gene; or homologs thereof.
 18. The method of claim 17,wherein the hasB gene is selected from the group consisting of (a) anucleic acid sequence encoding a polypeptide with an amino acid sequencehaving at least about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% identity to SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO:105; (b) a nucleic acid sequence which hybridizes under low, medium, orhigh stringency conditions with SEQ ID NO: 40, SEQ ID NO: 96, or SEQ IDNO: 104; and (c) a complementary strand of (a) or (b).
 19. The method ofclaim 18, wherein the hasB gene encodes a polypeptide having the aminoacid sequence of SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO: 105; or afragment thereof having UDP-glucose 6-dehydrogenase activity.
 20. Themethod of claim 17, wherein the tuaD gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 12; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 11; and (c) a complementary strandof (a) or (b).
 21. The method of claim 20, wherein the tuaD gene encodesa polypeptide having the amino acid sequence of SEQ ID NO: 12, or afragment thereof having UDP-glucose 6-dehydrogenase activity.
 22. Themethod of claim 16, wherein the UDP-glucose pyrophosphorylase gene is ahasC gene or gtaB gene; or homologs thereof.
 23. The method of claim 22,wherein the hasC gene is selected from the group consisting of (a) anucleic acid sequence encoding a polypeptide with an amino acid sequencehaving at least about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% identity to SEQ ID NO: 43, SEQ ID NO: 99, or SEQ ID NO:107; (b) a nucleic acid sequence which hybridizes under low, medium, orhigh stringency conditions with SEQ ID NO: 42 or SEQ ID NO: 98, or SEQID NO: 106; and (c) a complementary strand of (a) or (b).
 24. The methodof claim 23, wherein the hasC gene encodes a polypeptide having theamino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 99, or SEQ ID NO:107; or a fragment thereof having UDP-glucose pyrophosphorylaseactivity.
 25. The method of claim 22, wherein the gtaB gene is selectedfrom the group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 22; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 21; and (c) acomplementary strand of (a) or (b).
 26. The method of claim 25, whereinthe gtaB gene encodes a polypeptide having the amino acid sequence ofSEQ ID NO: 22, or a fragment thereof having UDP-glucosepyrophosphorylase activity.
 27. The method of claim 16, wherein theUDP-N-acetylglucosamine pyrophosphorylase gene is a hasD or gcaD gene;or homologs thereof.
 28. The method of claim 27, wherein the hasD geneis selected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 45; (b) a nucleic acid sequence which hybridizes underlow, medium, or high stringency conditions with SEQ ID NO: 44; and (c) acomplementary strand of (a) or (b).
 29. The method of claim 28, whereinthe hasD gene encodes a polypeptide having the amino acid sequence ofSEQ ID NO: 45, or a fragment thereof having UDP-N-acetylglucosaminepyrophosphorylase activity.
 30. The method of claim 27, wherein the gcaDgene is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 30; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 29; and (c) a complementary strand of (a) or (b).
 31. The method ofclaim 30, wherein the gcaD gene encodes a polypeptide having the aminoacid sequence of SEQ ID NO: 30, or a fragment thereof havingUDP-N-acetylglucosamine pyrophosphorylase activity.
 32. The method ofclaim 16, wherein the glucose-6-phosphate isomerase gene is a hasE orhomolog thereof.
 33. The method of claim 32, wherein the hasE gene isselected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 101; (b) a nucleic acid sequence which hybridizes underlow, medium, or high stringency conditions with SEQ ID NO: 100; and (c)a complementary strand of (a) or (b).
 34. The method of claim 33,wherein the hasE gene encodes a polypeptide having the amino acidsequence of SEQ ID NO: 101, or a fragment thereof havingglucose-6-phosphate isomerase activity.
 35. The method of claim 15,wherein the one or more genes encoding a precursor sugar is under thecontrol of the same or a different promoter(s) as the hyaluronansynthase encoding sequence.
 36. The method of claim 35, wherein the sameor the different promoter sequence comprises a “consensus” promoterhaving the sequence TTGACA for the “−35” region and TATAAT for the “−10”region.
 37. The method of claim 35, wherein the same or the differentpromoter sequence(s) is a tandem promoter in which each promoter of thetandem promoter is operably linked to the hyaluronan synthase encodingsequence.
 38. The method of claim 1, wherein the nucleic acid constructfurther comprises an mRNA processing/stabilizing sequence locateddownstream of the promoter sequence and upstream of the hyaluronansynthase encoding sequence.
 39. The method of claim 15, wherein thenucleic acid construct further comprises an mRNA processing/stabilizingsequence located downstream of a different promoter or differentpromoters of the one or more genes encoding enzymes in the biosynthesisof the precursor sugar and upstream of the one or more genes.
 40. Themethod of claim 1, wherein the nucleic acid construct further comprisesa selectable marker gene.
 41. The method of claim 1, wherein theBacillus host cell is selected from the group consisting of Bacillusagaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. 42.The method of claim 1, wherein the Bacillus host cell is Bacillussubtilis.
 43. The method of claim 1, wherein the Bacillus host cell isBacillus licheniformis.
 44. The method of claim 1, wherein the Bacillushost cell is unmarked with a selectable marker.
 45. The method of claim1, wherein the nucleic acid construct comprises a hyaluronan synthasegene, UDP-glucose 6-dehydrogenase gene, and UDP-glucosepyrophosphorylase gene operably linked to a short “consensus” promoterof amyQ having the sequence TTGAC for the “−35” region and TATAAT forthe “−10” region.
 46. A Bacillus host cell comprising a nucleic acidconstruct comprising a hyaluronan synthase encoding sequence operablylinked to a promoter sequence foreign to the hyaluronan synthaseencoding sequence.
 47. The Bacillus host cell of claim 46, wherein thehyaluronan synthase encoding sequence encodes a Group I hyaluronansynthase.
 48. The Bacillus host cell of claim 47, wherein the Group Ihyaluronan synthase encoding sequence is obtained from a Streptococcusstrain.
 49. The Bacillus host cell of claim 48, wherein theStreptococcus strain is Streptococcus equisimilis, Streptococcuspyogenes, Streptococcus uberis, or Streptococcus equi subsp.zooepidemicus.
 50. The Bacillus host cell of claim 46, wherein thehyaluronan synthase encoding sequence is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 2, SEQ ID NO:93, or SEQ ID NO: 103; (b) a nucleic acid sequence which hybridizesunder low, medium, or high stringency conditions with SEQ ID NO: 1, SEQID NO: 92, or SEQ ID NO: 102; and (c) a complementary strand of (a) or(b).
 51. The Bacillus host cell of claim 50, wherein the hyaluronansynthase encoding sequence encodes a polypeptide with the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; or afragment thereof encoding a polypeptide having hyaluronan synthaseactivity.
 52. The Bacillus host cell of claim 46, wherein the hyaluronansynthase encoding sequence encodes a Group II hyaluronan synthase. 53.The Bacillus host cell of claim 52, wherein the Group II hyaluronansynthase encoding sequence is obtained from a Pasteurella strain. 54.The Bacillus host cell of claim 53, wherein the Pasteurella strain isPasteurella multocida.
 55. The Bacillus host cell of claim 46, whereinthe hyaluronan synthase encoding sequence is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 95; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 94; and (c) a complementary strandof (a) or (b).
 56. The Bacillus host cell of claim 55, wherein thehyaluronan synthase encoding sequence encodes a polypeptide with theamino acid sequence of SEQ ID NO: 95, or a fragment thereof encoding apolypeptide having hyaluronan synthase activity.
 57. The Bacillus hostcell of claim 46, wherein a precursor sugar of the hyaluronic acid issupplied to or produced by the Bacillus host cell.
 58. The Bacillus hostcell of claim 57, wherein the precursor sugar is D-glucuronic acid orN-acetyl-glucosamine.
 59. The Bacillus host cell of claim 57, whereinthe precursor sugar is encoded by endogenous genes, by non-endogenousgenes, or by a combination of endogenous and non-endogenous genes in theBacillus host cell.
 60. The Bacillus host cell of claim 46, wherein thenucleic acid construct further comprises one or more genes encodingenzymes in the biosynthesis of a precursor sugar of the hyaluronic acidor the Bacillus host cell comprises one or more second nucleic acidconstructs comprising one or more genes encoding enzymes in thebiosynthesis of a precursor sugar of the hyaluronic acid.
 61. TheBacillus host cell of claim 60, wherein the one or more genes isselected from the group consisting of a UDP-glucose 6-dehydrogenasegene, UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene.
 62. The Bacillus host cell of claim 61, whereinthe UDP-glucose 6-dehydrogenase gene is a hasB gene or tuaD gene; orhomologs thereof.
 63. The Bacillus host cell of claim 62, wherein thehasB gene is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO: 105; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 40, SEQ ID NO: 96, or SEQ ID NO:104; and (c) a complementary strand of (a) or (b).
 64. The Bacillus hostcell of claim 63, wherein the hasB gene encodes a polypeptide having theamino acid sequence SEQ ID NO: 41, SEQ ID NO: 97, or SEQ ID NO: 105; ora fragment thereof having UDP-glucose 6-dehydrogenase activity.
 65. TheBacillus host cell of claim 62, wherein the tuaD gene is selected fromthe group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 12; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 11; and (c) acomplementary strand of (a) or (b).
 66. The Bacillus host cell of claim65, wherein the tuaD gene encodes a polypeptide having the amino acidsequence SEQ ID NO: 12, or a fragment thereof having UDP-glucose6-dehydrogenase activity.
 67. The Bacillus host cell of claim 61,wherein the UDP-glucose pyrophosphorylase gene is a hasC gene or gtaBgene; or homologs thereof.
 68. The Bacillus host cell of claim 67,wherein the hasC gene is selected from the group consisting of (a) anucleic acid sequence encoding a polypeptide with an amino acid sequencehaving at least about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% identity to SEQ ID NO: 43, SEQ ID NO: 99, or SEQ ID NO:107; (b) a nucleic acid sequence which hybridizes under low, medium, orhigh stringency conditions with SEQ ID NO: 42 or SEQ ID NO: 98, or SEQID NO: 106; and (c) a complementary strand of (a) or (b).
 69. TheBacillus host cell of claim 68, wherein the hasC gene encodes apolypeptide having the amino acid sequence SEQ ID NO: 43, SEQ ID NO: 99,or SEQ ID NO: 107; or a fragment thereof having UDP-glucosepyrophosphorylase activity.
 70. The Bacillus host cell of claim 61,wherein the gtaB gene is selected from the group consisting of (a) anucleic acid sequence encoding a polypeptide with an amino acid sequencehaving at least about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% identity to SEQ ID NO: 22; (b) a nucleic acid sequencewhich hybridizes under low, medium, or high stringency conditions withSEQ ID NO: 21; and (c) a complementary strand of (a) or (b).
 71. TheBacillus host cell of claim 70, wherein the gtaB gene encodes apolypeptide having the amino acid sequence SEQ ID NO: 22, or a fragmentthereof having UDP-glucose pyrophosphorylase activity.
 72. The Bacillushost cell of claim 61, wherein the UDP-N-acetylglucosaminepyrophosphorylase gene is a hasD or gcaD gene; or homologs thereof. 73.The Bacillus host cell of claim 72, wherein the hasD gene is selectedfrom the group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 45; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 44; and (c) acomplementary strand of (a) or (b).
 74. The Bacillus host cell of claim73, wherein the hasD gene encodes a polypeptide having the amino acidsequence SEQ ID NO: 45, or a fragment thereof havingUDP-N-acetylglucosamine pyrophosphorylase activity.
 75. The Bacillushost cell of claim 72, wherein the gcaD gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%identity to SEQ ID NO: 30; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 29; and (c) a complementary strandof (a) or (b).
 76. The Bacillus host cell of claim 75, wherein the gcaDgene encodes a polypeptide having the amino acid sequence of SEQ ID NO:30, or a fragment thereof having UDP-N-acetylglucosaminepyrophosphorylase activity.
 77. The Bacillus host cell of claim 61,wherein the glucose-6-phosphate isomerase gene is a hasE or homologthereof.
 78. The Bacillus host cell of claim 77, wherein the hasE geneis selected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 101; (b) a nucleic acid sequence which hybridizes underlow, medium, or high stringency conditions with SEQ ID NO: 100; and (c)a complementary strand of (a) or (b).
 79. The Bacillus host cell ofclaim 78, wherein the hasE gene encodes a polypeptide having the aminoacid sequence SEQ ID NO: 101, or a fragment thereof havingglucose-6-phosphate isomerase activity.
 80. The Bacillus host cell ofclaim 60, wherein the one or more genes encoding a precursor sugar areunder the control of the same or a different promoter(s) as thehyaluronan synthase encoding sequence.
 81. The Bacillus host cell ofclaim 80, wherein the same or the different promoter sequence(s)comprises a “consensus” promoter having the sequence TTGACA for the“-35” region and TATAAT for the “−10” region.
 82. The Bacillus host cellof claim 80, wherein the same or the different promoter or the differentpromoters is a tandem promoter in which each promoter of the tandempromoter is operably linked to the hyaluronan synthase encodingsequence.
 83. The Bacillus host cell of claim 46, wherein the nucleicacid construct further comprises an mRNA processing/stabilizing sequencelocated downstream of the promoter sequence and upstream of thehyaluronan synthase encoding sequence.
 84. The Bacillus host cell ofclaim 60, wherein the nucleic acid construct further comprises an mRNAprocessing/stabilizing sequence located downstream of the differentpromoter sequence(s) of the one or more genes encoding enzymes in thebiosynthesis of a precursor sugar of the hyaluronic acid and upstream ofthe one or more genes.
 85. The Bacillus host cell of claim 46, whereinthe nucleic acid construct further comprises a selectable marker gene.86. The Bacillus host cell of claim 47, wherein the Bacillus host cellis selected from the group consisting of Bacillus agaradherens, Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus firmus,Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillussubtilis, and Bacillus thuringiensis.
 87. The Bacillus host cell ofclaim 46, wherein the Bacillus host cell is Bacillus subtilis.
 88. TheBacillus host cell of claim 46, wherein the Bacillus host cell isBacillus licheniformis.
 89. The Bacillus host cell of claim 46, which isunmarked with a selectable marker.
 90. The Bacillus host cell of claim46, wherein the nucleic acid construct comprises a hyaluronan synthasegene, UDP-glucose 6-dehydrogenase gene, and UDP-glucosepyrophosphorylase gene operably linked to a short “consensus” promoterof amyQ having the sequence TTGACA for the “−35” region and TATAAT forthe “−10” region.
 91. A nucleic acid construct comprising a hyaluronansynthase encoding sequence operably linked to a promoter sequenceforeign to the hyaluronan synthase encoding sequence.
 92. The constructof claim 91, wherein the gene for precursor sugar is expressed from thesame promoter as the promoter of the hyaluronan synthase encodingsequence.
 93. The construct of claim 91, wherein the gene for precursorsugar is expressed from a different promoter as the promoter of thehyaluronan synthase encoding sequence.
 94. The construct of claim 91,wherein the hyaluronan synthase encoding sequence encodes a Group Ihyaluronan synthase.
 95. The construct of claim 94, wherein the Group Ihyaluronan synthase encoding sequence is obtained from a Streptococcusstrain.
 96. The construct of claim 95, wherein the Streptococcus strainis Streptococcus equisimilis, Streptococcus pyogenes, Streptococcusuberis, or Streptococcus equi subsp. zooepidemicus.
 97. The construct ofclaim 91, wherein the hyaluronan synthase encoding sequence is selectedfrom the group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; (b) a nucleic acid sequencewhich hybridizes under low, medium, or high stringency conditions withSEQ ID NO: 1, SEQ ID NO: 92, or SEQ ID NO: 102; and (c) a complementarystrand of (a) or (b).
 98. The construct of claim 97, wherein thehyaluronan synthase encoding sequence encodes a polypeptide having theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103;or a fragment thereof having hyaluronan synthase activity.
 99. Theconstruct of claim 91, wherein the hyaluronan synthase encoding sequenceencodes a Group II hyaluronan synthase.
 100. The construct of claim 99,wherein the Group II hyaluronan synthase encoding sequence is obtainedfrom a Pasteurella strain.
 101. The construct of claim 100, wherein thePasteurella strain is Pasteurella multocida.
 102. The construct of claim91, wherein the hyaluronan synthase encoding sequence is selected fromthe group consisting of (a) a nucleic acid sequence encoding apolypeptide with an amino acid sequence having at least about 70%, about75%, about 80%, about 85%, about 90%, or about 95% identity to SEQ IDNO: 95; (b) a nucleic acid sequence which hybridizes under low, medium,or high stringency conditions with SEQ ID NO: 94; and (c) acomplementary strand of (a) or (b).
 103. The construct of claim 102,wherein the hyaluronan synthase encoding sequence encodes a polypeptidehaving the amino acid sequence of SEQ ID NO: 95, or a fragment thereofhaving hyaluronan synthase activity.
 104. The construct of claim 91,wherein the nucleic acid construct further comprises one or more genesencoding enzymes in the biosynthesis of a precursor sugar of thehyaluronic acid or the Bacillus host cell further comprises one or moresecond nucleic acid constructs comprising one or more genes encodingenzymes in the biosynthesis of a precursor sugar of the hyaluronic acid.105. The construct of claim 104, wherein the one or more genes isselected from the group consisting of a UDP-glucose 6-dehydrogenasegene, UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene.
 106. The construct of claim 105, wherein theUDP-glucose 6-dehydrogenase gene is a hasB gene or tuaD gene; orhomologs thereof.
 107. The construct of claim 106, wherein the hasB geneis selected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 97 or SEQ ID NO: 99; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 96 or SEQ ID NO: 98; and (c) a complementary strand of (a) or (b).108. The construct of claim 107, wherein the hasB gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 97 or SEQ IDNO: 99, or a fragment thereof having UDP-glucose 6-dehydrogenaseactivity.
 109. The construct of claim 106, wherein the tuaD gene isselected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 12; (b) a nucleic acid sequence which hybridizes underlow, medium, or high stringency conditions with SEQ ID NO: 11; and (c) acomplementary strand of (a) or (b).
 110. The construct of claim 109,wherein the tuaD gene encodes a polypeptide having the amino acidsequence of SEQ ID NO: 12, or a fragment thereof having UDP-glucose6-dehydrogenase activity.
 111. The construct of claim 105, wherein theUDP-glucose pyrophosphorylase gene is a hasC gene or gtaB gene; orhomologs thereof.
 112. The construct of claim 111, wherein the hasC geneis selected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 43, SEQ ID NO: 99, or SEQ ID NO: 107; (b) a nucleic acidsequence which hybridizes under low, medium, or high stringencyconditions with SEQ ID NO: 42 or SEQ ID NO: 98, or SEQ ID NO: 106; and(c) a complementary strand of (a) or (b).
 113. The construct of claim112, wherein the hasC gene encodes a polypeptide having the amino acidsequence of SEQ ID NO: 43, SEQ ID NO: 99, or SEQ ID NO: 107; or afragment thereof having UDP-glucose pyrophosphorylase activity.
 114. Theconstruct of claim 111, wherein the gtaB gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ is ID NO: 22; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 21; and (c) a complementary strandof (a) or (b).
 115. The construct of claim 1124, wherein the gtaB geneencodes a polypeptide having the amino acid sequence of SEQ ID NO: 22,or a fragment thereof having UDP-glucose pyrophosphorylase activity.116. The construct of claim 105, wherein the UDP-N-acetylglucosaminepyrophosphorylase gene is a hasD or gcaD gene; or homologs thereof. 117.The construct of claim 116, wherein the hasD gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 105; (b)a nucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 104; and (c) a complementarystrand of (a) or (b).
 118. The construct of claim 117, wherein the hasDgene encodes a polypeptide having the amino acid sequence of SEQ ID NO:105, or a fragment thereof having UDP-N-acetylglucosaminepyrophosphorylase activity.
 119. The construct of claim 116, wherein thegcaD gene is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 30; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 29; and (c) a complementary strand of (a) or (b).
 120. The constructof claim 119, wherein the gcaD gene encodes a polypeptide having theamino acid sequence of SEQ ID NO: 30, or a fragment thereof havingUDP-N-acetylglucosamine pyrophosphorylase activity.
 121. The constructof claim 105, wherein the glucose-6-phosphate isomerase gene is a hasEor homolog thereof.
 122. The construct of claim 121, wherein the hasEgene is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 101; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 100; and (c) a complementary strand of (a) or (b).
 123. Theconstruct of claim 122, wherein the hasE gene encodes a polypeptidehaving the amino acid sequence SEQ ID NO: 101, or a fragment thereofhaving glucose-6-phosphate isomerase activity.
 124. The construct ofclaim 104, wherein the one or more genes encoding a precursor sugar isunder the control of the same or a different promoter or differentpromoters as the hyaluronan synthase encoding sequence.
 125. Theconstruct of claim 124, wherein the same or the different promoter(s)comprises a “consensus” promoter having the sequence TTGACA for the“−35” region and TATAAT for the “31 10” region.
 126. The construct ofclaim 130, wherein the same or the different promoter sequence is atandem promoter in which each promoter of the tandem promoter isoperably linked to the hyaluronan synthase encoding sequence.
 127. Theconstruct of claim 91, which further comprises an mRNAprocessing/stabilizing sequence located downstream of the promotersequence and upstream of the hyaluronan synthase encoding sequence. 128.The construct of claim 124, which further comprises an mRNAprocessing/stabilizing sequence located downstream of the differentpromoter or each of the different promoters of the one or more genesencoding enzymes in the biosynthesis of a precursor sugar of thehyaluronan and upstream of the one or more genes.
 129. The construct ofclaim 91, which further comprises a selectable marker gene.
 130. Theconstruct of claim 91, which comprises a hyaluronan synthase gene,UDP-glucose 6-dehydrogenase gene, and UDP-glucose pyrophosphorylase geneoperably linked to a short “consensus” promoter of amyQ having thesequence TTGACA for the “−35” region and TATAAT for the “−10” region.131. An isolated nucleic acid sequence encoding a hyaluronan synthaseoperon comprising a hyaluronan synthase gene or portion thereof and aUDP-glucose 6-dehydrogenase gene, and optionally one or more genesselected from the group consisting of a UDP-glucose pyrophosphorylasegene, UDP-N-acetylglucosamine pyrophosphorylase gene, andglucose-6-phosphate isomerase gene.
 132. The isolated nucleic acidsequence of claim 131, wherein the hyaluronan synthase gene is SEQ IDNO: 1, SEQ ID NO: 92, SEQ ID NO: 94, or SEQ ID NO: 102; or fragmentsthereof that encode a polypeptide having hyaluronan synthase activity.133. The isolated nucleic acid sequence of claim 131, wherein theUDP-glucose 6-dehydrogenase gene is SEQ ID NO: 11, SEQ ID NO: 40, or SEQID NO: 96, or SEQ ID NO: 104; or fragments thereof that encode apolypeptide having UDP-glucose 6-dehydrogenase activity.
 134. Theisolated nucleic acid sequence of claim 131, wherein the UDP-glucosepyrophosphorylase gene is SEQ ID NO: 21, SEQ ID NO: 42, or SEQ ID NO:98, SEQ ID NO: 106; or fragments thereof that encode a polypeptidehaving UDP-glucose pyrophosphorylase activity.
 135. The isolated nucleicacid sequence of claim 131, wherein the UDP-N-acetylglucosaminepyrophosphorylase gene is SEQ ID NO: 29 or SEQ ID NO: 44; or fragmentsthereof that encode a polypeptide having UDP-N-acetylglucosaminepyrophosphorylase activity.
 136. The isolated nucleic acid sequence ofclaim 131, wherein the glucose-6-phosphate isomerase gene is SEQ ID NO:100; or fragments thereof that encode a polypeptide havingglucose-6-phosphate isomerase activity.
 137. The isolated nucleic acidsequence of claim 131, which further comprises one or more genesselected from the group consisting of a hexokinase gene,phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyltransferase gene.
 138. The isolated nucleic acid sequence of claim 131,which comprises a hyaluronan synthase gene, UDP-glucose 6-dehydrogenasegene, and UDP-glucose pyrophosphorylase gene.
 139. The isolated nucleicacid sequence of claim 131, having the nucleic acid sequence of SEQ IDNO:
 108. 140. A nucleic acid construct comprising the nucleic acidsequence of claim
 131. 141. An expression vector comprising the nucleicacid construct of claim
 139. 142. An isolated nucleic acid sequenceencoding a UDP-glucose 6-dehydrogenase selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least about 75%, about 80%, about85%, about 90%, or about 95% identity to SEQ ID NO: 41; (b) a nucleicacid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to SEQID NO: 40; (c) a nucleic acid sequence which hybridizes under medium orhigh stringency conditions with (i) the nucleic acid sequence of SEQ IDNO: 40, (ii) the cDNA sequence contained in SEQ ID NO: 40, or (iii) acomplementary strand of (i) or (ii); and (d) a subsequence of (a), (b),or (c), wherein the subsequence encodes a polypeptide fragment which hasUDP-glucose 6-dehydrogenase activity.
 143. The nucleic acid sequence ofclaim 142, which encodes a polypeptide having an amino acid sequencewhich has at least about 75%, about 80%, about 85%, about 90%, or about95% identity the amino acid sequence of SEQ ID NO:
 41. 144. The nucleicacid sequence of claim 142, which encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:
 41. 145. The nucleic acid sequence ofclaim 142, which encodes a polypeptide consisting of the amino acidsequence of SEQ ID NO: 41, or a fragment thereof which has UDP-glucose6-dehydrogenase activity.
 146. The nucleic acid sequence of claim 145,which encodes a polypeptide consisting of the amino acid sequence of SEQID NO:
 41. 147. The nucleic acid sequence of claim 142, which has thenucleic acid sequence of SEQ ID NO:
 40. 148. The nucleic acid sequenceof claim 142, wherein the nucleic acid sequence hybridizes under mediumor high stringency conditions with (i) the nucleic acid sequence of SEQID NO: 40, (ii) the cDNA sequence contained in SEQ ID NO: 40, or (iii) acomplementary strand of (i) or (ii).
 149. The nucleic acid sequence ofclaim 142, which is contained in the plasmid pMRT106 which is containedin Escherichia coli NRRL B-30536.
 150. A nucleic acid constructcomprising the nucleic acid sequence of claim 142 operably linked to oneor more control sequences which direct the production of the polypeptidein a suitabe expression host.
 151. A recombinant expression vectorcomprising the nucleic acid construct of claim
 150. 152. A recombinanthost cell comprising the nucleic acid construct of claim
 150. 153. Amethod for producing a polypeptide having UDP-glucose 6-dehydrogenaseactivity comprising (a) cultivating the host cell of claim 152 underconditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 154. An isolated polypeptide havingUDP-glucose 6-dehydrogenase activity encoded by the nucleic acidsequence of claim
 142. 155. An isolated nucleic acid sequence encoding aUDP-glucose pyrophosphorylase selected from the group consisting of: (a)a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least about 90%, 95%, or 97% identity to SEQ IDNO: 43; (b) a nucleic acid sequence having at least about 90%, about95%, or about 97% homology to SEQ ID NO: 42; (c) a nucleic acid sequencewhich hybridizes under high or very high stringency conditions with (i)the nucleic acid sequence of SEQ ID NO: 42, (ii) the cDNA sequencecontained in SEQ ID NO: 42, or (iii) a complementary strand of (i) or(ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequenceencodes a polypeptide fragment which has UDP-glucose pyrophosphorylaseactivity.
 156. The nucleic acid sequence of claim 155, which encodes apolypeptide having an amino acid sequence which has at least about 90%,about 95%, or about 97% identity the amino acid sequence of SEQ ID NO:43.
 157. The nucleic acid sequence of claim 155, which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:
 43. 158.The nucleic acid sequence of claim 155, which encodes a polypeptideconsisting of the amino acid sequence of SEQ ID NO: 43, or a fragmentthereof which has UDP-glucose pyrophosphorylase activity.
 159. Thenucleic acid sequence of claim 158, which encodes a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 43. 160. The nucleicacid sequence of claim 155, which has the nucleic acid sequence of SEQID NO:
 42. 161. The nucleic acid sequence of claim 155, wherein thenucleic acid sequence hybridizes under high stringency or very highconditions with (i) the nucleic acid sequence of SEQ ID NO: 42, (ii) thecDNA sequence contained in SEQ ID NO: 42, or (iii) a complementarystrand of (i) or (ii).
 162. The nucleic acid sequence of claim 155,which is contained in the plasmid pMRT106 which is contained inEscherichia coli NRRL B-30536.
 163. A nucleic acid construct comprisingthe nucleic acid sequence of claim 155 operably linked to one or morecontrol sequences which direct the production of the polypeptide in asuitable expression host.
 164. A recombinant expression vectorcomprising the nucleic acid construct of claim
 163. 165. A recombinanthost cell comprising the nucleic acid construct of claim
 163. 166. Amethod for producing a polypeptide having UDP-glucose pyrophosphorylaseactivity comprising (a) cultivating the host cell of claim 165 underconditions suitable for production of the polypeptide; and (b)recovering the polypeptide.
 167. An isolated polypeptide havingUDP-glucose 6-dehydrogenase activity encoded by the nucleic acidsequence of claim
 155. 168. An isolated nucleic acid sequence encoding aUDP-N-acetylglucosamine pyrophosphorylase selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least about 75%, about 80%, about85%, about 90%, or about 95% identity to SEQ ID NO: 45; (b) a nucleicacid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to SEQID NO: 44; (c) a nucleic acid sequence which hybridizes under low,medium, or high stringency conditions with (i) the nucleic acid sequenceof SEQ ID NO: 44, (ii) the cDNA sequence contained in SEQ ID NO: 44, or(iii) a complementary strand of (i) or (ii); and (d) a subsequence of(a), (b), or (c), wherein the subsequence encodes a polypeptide fragmentwhich has UDP-N-acetylglucosamine pyrophosphorylase activity.
 169. Thenucleic acid sequence of claim 168, which encodes a polypeptide havingan amino acid sequence which has at least about 75%, about 80%, about85%, about 90%, or about 95% identity the amino acid sequence of SEQ IDNO:
 45. 170. The nucleic acid sequence of claim 168, which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:
 45. 171.The nucleic acid sequence of claim 168, which encodes a polypeptideconsisting of the amino acid sequence of SEQ ID NO: 45, or a fragmentthereof which has UDP-N-acetylglucosamine pyrophosphorylase activity.172. The nucleic acid sequence of claim 171, which encodes a polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 45. 173. The nucleicacid sequence of claim 168, which has the nucleic acid sequence of SEQID NO:
 44. 174. The nucleic acid sequence of claim 168, wherein thenucleic acid sequence hybridizes under low, medium, or high stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 44, (ii) thecDNA sequence contained in SEQ ID NO: 44, or (iii) a complementarystrand of (i) or (ii).
 175. The nucleic acid sequence of claim 168,which is contained in the plasmid pMRT106 which is contained inEscherichia coli NRRL B-30536.
 176. A nucleic acid construct comprisingthe nucleic acid sequence of claim 168 operably linked to one or morecontrol sequences which direct the production of the polypeptide in asuitable expression host.
 177. A recombinant expression vectorcomprising the nucleic acid construct of claim
 176. 178. A recombinanthost cell comprising the nucleic acid construct of claim
 176. 179. Amethod for producing a polypeptide having UDP-N-acetylglucosaminepyrophosphorylase activity comprising (a) cultivating the host cell ofclaim 178 under conditions suitable for production of the polypeptide;and (b) recovering the polypeptide.
 180. An isolated polypeptide havingUDP-glucose 6-dehydrogenase activity encoded by the nucleic acidsequence of claim
 168. 181. An isolated polypeptide having UDP-glucose6-dehydrogenase activity encoded by the nucleic acid sequence of claim168.
 182. The method of claim 1, wherein the Bacillus host cell containsa disrupted or deleted cypX and/or yvmC gene.
 183. The Bacillus hostcell of claim 46, which contains a disrupted or deleted cypX and/or yvmCgene.